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Cell Proliferation - Science topic

Cell Proliferation is an all of the processes involved in increasing CELL NUMBER including CELL DIVISION.
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I have generated cell proliferation luminescences OD value of breast cancer cells. Now, I am trying to calculate CI value with compusyn software. Can anyone help me to convert OD into effect as compusyn software only takes a value of 0-1 but my OD values are in hundred to thousand range.
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Why are you using OD values for luminescence? It seems as if you are using film OD for luminescence and thus risk saturation effect problems. I suggest that you use the actual light emitted (luminescence) values and, after subtracting appropriate blank background, and assuring that the luminescence of each cell is equal, take the ratios on the growing cells mean value at each time point relative to that of the original day zero value (use equipartition of variance to get the appropriate precision estimates of the ratios) and plot their values on a semilog plot as a surrogate of cell growth, which is what I assume that you are seeking to measure and compare.
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Hi All,
My team and I have been growing endometrial organoids and recently noticed a strange cell growth appearing in one of our plates, what appears to be a cluster of cells with spindle like formations growing outward. It is too early to tell how exactly the organoid's growth is being affected, however is does appear cell proliferation has slowed since these growths were noticed. Some members of our lab have suggested yeast or other types of bacterial contamination, or fungus. We on the fence about what to do, since our cell lines are precious and relatively difficult to obtain, throwing out all the cells would be a great loss. However, if you think it was a fungal or bacterial contamination that could create spores or in other ways endanger specimens in our incubator, we would dispose of immediately. Thank you very much for your time, and advice.
Best wishes,
Etta Hanlon
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Hi Etta, I have been culturing chicken intestine organoids in BME matrix and I've seen spindle cells develop from organoids after 24h of culture. We think they are fibroblasts. Did you solve this problem?
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Why do reagents like the ones below induce lytic replication of Epstein-Barr virus (EBV)?
What's the biochemical explanation?
Is this something specific to EBV?
* 12-O-tetradecanoylphorbol-13-acetate (TPA)
* 5-aza-deoxycytidine (5-aza)
* Calcium ionophore
* Sodium butyrate
* Tetracycline derivative doxycycline (Dox)
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EBV seems to employ epigenetic mechanisms to control the host transcriptome and to control expression of its own virally encoded genes. Upon initial infection of a cell, the unmethylated viral genome can undergo viral replication with new virion production, while a subset of infected cells acquires a highly methylated viral genome that squelches expression of foreign proteins and mediates long-term viral persistence by way of latent infection. Infected tumors tend to have highly methylated EBV DNA, and methylation-related silencing of viral genes helps explain how infected tumors evade immune destruction.
The reagents mentioned by you like 5-aza-deoxycytidine (5-aza), Sodium butyrate, Tetracycline derivative doxycycline (Dox), etc. are demethylating agents with the capacity to induce transient DNA hypomethylation.
Deliberate attempt to reduce the DNA methylation in EBV-associated cancer with the aim to activate the expression of cellular and viral genes has the potential to achieve two potentially therapeutic events.
First, it may result in reactivation of the expression of viral genes that expose the cells to attack by the immune system (the recognition of the target by virus-specific cytotoxic T-cells).
Secondly, it may result in the expression of important cellular genes that cause the cancer cells to stop proliferating or to die through apoptosis.
So, deliberate attempt to alter DNA methylation presents an attractive research direction which may lead to new therapeutic approaches to treat EBV-associated cancer.
In a way, yes, it is specific to EBV because EBV employs epigenetic mechanisms namely, attains a highly methylated viral genome that helps mediate long-term viral persistence by way of latent infection evading the immune system. When demethylating agents are used, they reactivate viral gene expression and as a result are easily recognized and attacked by the immune system.
I have attached a research article for your reference.
Best Wishes.
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I did the Vero cell cultivation at different cell seeding density for toxicity effect study, and in the last two sets of MTT assays, cell died after MTT addition (even 10 minutes after, I just took a picture after 10 minutes and the cells were starting to detach from the surface, cell sheets detachment!).
In the first experiment, I had 3 different cell inoculation density in which I have the cell density range of low and high, but after MTT addition, they were all dead.
And at the second time, due to MTT reagent cytotoxic effect, instead of using 1:3 ratio of MTT solution, I used 1:6 ratio of MTT solution and the medium (I am using Cell Proliferation Kit II (XTT) from Roche)
I would appreciate any ideas or suggestions on what might be the reason for this.
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Hi, I'm finishing up my PhD in a venom analysis lab. I do massive amounts of toxicity, cytotoxicity, toxic mechanisms, differential and selective toxicity, and use everything from counting cells by eye, by trypan blue stain using a Countess, by various flow cytometric assays, and my PI has been at the forefront of venom analysis for decades. When I am contracted by pharmaceutical companies trying to develop a drug they prefer MTT assays as their initial "cytotoxic or not" and "approximate EC50 dose". Even though it's indirect it is far easier to run multiple replicates of multiple doses even when you have little material (such as an isolated scorpion venom protein... it takes a lot of, potentially hazardous, work to get a few micrograms sometimes). So I am intimately familiar with MTT and (if you haven't looked into this, I recommend it) MTS.
As for why you are seeing cells die after the addition of even small amounts of MTT it is because MTT is extremely cytotoxic to any cell that produces NADH (so... cells that do cellular respiration) is that MTT kills cells. "Intracellular metabolism of MTT has been shown to gradually cause mitochondrial injury, disturbance of normal cell metabolism, and finally cell apoptosis [4,41]. Time-dependent loss of membrane integrity as a result of formazan exocytosis has also been proposed as a mechanism of cell death following MTT incubation" from
I don't think you pipetted too hard. You need to titurate cells well following MTT treatment to get a good result and you need to solubilize the cells with DMSO or another detergent. Formazan (reduced MTT) forms insoluble crystals, you can see these after treatment with MTT, these have been shown to build up in multiple organelles including mitochondria, ribosomes, nucleus, and not only interfere with cellular operations but can pierce and lyse cellular membranes in a similar manner to any crystal forming within a cell (such as ice). Even in extremely low doses I think you would witness apoptosis induction in healthy cells, and the loss of adhesion due to loss of membrane integrity and effective protein synthesis and the consumption of the NADH necessary to maintain vaiability. MTT is not an assay that is meant to result in intact and definitely not living cells. It is essentially quantifying the amount of reducing agent present (which is typically correlated to cell number, but it varies for every cell type and every treatment and you should always have an identical treatment ladder with varying cell numbers ranging from zero to double what your treatment wells will be seeded with). The nice part of this is that I have found that incubating my cells with MTT overnight results in a far more consistent result, and the use of a ladder as described below demonstrates whether the cell numbers used, incubation time, MTT dose, and carrier provided a relationship between seeded number of cells and optical absorbance that has (for my purposes) an R2 value of over .9 for either logarithmic, asymptotic, or linear best fit lines. It's also acceptable, in my opinion, to exclude the double population and lowest population if these are outside a clearly linear, with high R2 value, relationship for all other cell numbers and absorbances. However, this also means that you should not be including any treatments where the results were outside your new maximum and minimum (the lowest viability would be the lowest population you included in you best fit defining data from the ladder). This is also typically only useful in the case of analyzing cytotoxins by dose and trying to find the dose that results in an equal absorbance as if you simply seeded the wells with half the cells, since if you have 100%, 50%, 25% and 12.5 % but 200% and 6.125% were not within the linear range it doesn't mean you need to throw out all your data... most treatments will be somewhere between 100% and 12.5% relative viability anyway... assuming there was some time that the cells were alive and produced some NADH then even only 1 hour should have been eough time for the 100% population to produce as much as a population 1/24th it's size if incubated for 1 day.
Anyway, I just wanted to elaborate a bit and explain why I am of the opinion that trying to keep your cells alive after exposure to MTT is an unlikely task. MTS is water soluble and does not form crystals and it may be possible to find a dose that is both observable colorimetrically and may not be completely wiping out the populations... but for MTT I don't think you would get surviving cells if you've added a dose that is useful for quantifying things at any level.
. If you don't use a ladder (every single plate i use for an MTT assay has 4 columns devoted to a ladder, the first containing double the cell population as my treatment wells, the second identical, the third half my treatment, then a quarter, then an eighth, and finally a media only and a carrier +media only, this ladder should result (with 4 reps at each population) in relatively consistent results (they should be seeded at the same time as the rest of your wells and treated identically to your control wells, same volume of MTT, same incubation, read at the same time... it's on the same plate so this shouldn't be difficult to make the conditions close to identical except for position on the plate... so remember that corners and edges evaporate faster and will often seem slightly darker and more absorbant as the same quantity of f
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Hello,
I`m testing LPS (10 ug/mL to 40 ug/mL) with IEC-18 cells and doesn´t reduce cell proliferation (MTT assay).
We have changed the cell type and tested several batches of LPS and we don't know what is going on.
Could you tell me what reference you have used from LPS and has it worked for you?
Or what could be the problem?
Thank you!!
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I have some experience with IEC-18 cells and inducing several toxins including LPS. Hence, I would say the toxicity of LPS in IEC-18 is comparatively lower (RAW264.7, BV-2, and so on).
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In my cell culture i have GFP positive and negative cells. I would like to know if there is a difference in cell proliferation rate in positive compared to negative cells without sorting the cells.
Normally we just count the cells to find out the proliferation rate but i have a mixture of positive and negative cells. I would like to compare the two. Could i use FACS somehow to gate the positive cells and find out if the cell went through more dividing cycles as the negative control?
Any suggestions?
I have found 3H-thymidine, but after reading a paper it seems quite unreliable.
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As mentioned above, i can't sort the cells. It has to be done with the mixture of cells.
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It is an experiment to check the effect of a certain phytoestrogen on a particular cancer cell proliferation in-vitro.
The results consists of
Six absorption readings from control group
Six absorption readings from X phytoestrogen at concentration A
Six absorption readings from Y phytoestrogen at concentration a
Six absorption readings from Y phytoestrogen at concentration b
Six absorption readings from Y phytoestrogen at concentration c
THANK YOU.
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Hello John Kurian,
Since you are testing two phytoestrogens (X and Y), out of which Y phytoestrogen has three concentrations (a,b,c). So you have to see the effects of two phytoestrogens, therefore it would be better to use two-way ANOVA followed by post hoc analysis (to compare any two groups). Actually, in your experiment, you have first factor = X phytoestrogen; second factor =Y phytoestrogen and then interaction between these two factors X vs Y (third factor).
If you want to only analyze the effect of various concentrations (a,b,c) of Y phytoestrogen (one factor) , then one-way ANOVA followed by post hoc analysis would be appropriate.
Hope this helps
Best
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Dear all,
Recently I met a problem. We screened out a gene A which performs like a tumor suppressor gene. It negatively correlates with clinical patients' survival. And it affectes tumor cell proliferation much. When we knockdowm it the proliferation of lung tumor cell line increases. The radiosensitivity seemed to be decreased. My quenstion is: Commonly what should the radiosensitivity be implicated when the proliferation was upregulated by one gene? If my results are repeatable, is it strange as we inhibit a tumor suppressor gene to achieve radiosensitivity? I read some papers about the relationship between cell proliferation and its radiosensitivity, no consensus opinion was found. What do you think about this kind of thing?
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What Malcom refers to is the "Law of Bergonie & Tribondeau". This is misunderstood in the Anglophone literature, due to a mistranslation (from their French) of their reported observations. A more correct translation of their reported "appear to be more radiosensitive" is not the "are more radiosensitive" as translated in Radiation Research. What best explains the B&T observations is that a cell that has been sterilized (lost it's reproductive integrity or clonogenicity) will not be seen to be sterilized until it attempts to divide (mitosis) and fails (most apoptosis in solid tissues occurs after a failed mitosis). This is best exemplified in lethally irradiated hibernating squirrels, which are very radioresistant compared to active squirrels, but once brought out of hibernation, die with the same time course post-hibernation as that observed for normally active squirrels post-irradiation (see CS Lange, DPhil thesis, Oxford University, Fac Med, Bodleian Library, 1968). Hence, radiosensitivity should be measured in terms of loss of reproductive integrity (ability to proliferate ad infinitum, or clonogenicity, where a sufficiently large number of divisions (at least 10) are taken as the endpoint).
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Because I want to make B16F10 cells proliferate faster in vitro, I wonder if 20%FBS will be better. Thank you.
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Increasing the % of FBS would certainly help in accelerating the proliferation of your cells. But, it may also have unexpected outcomes when it comes to cell fitness and immunoregulatory activities. But such cells should not be preferred for regular experiments as they may lead to some circumstantial artifacts and unwarranted outcomes. I think it would be more appropriate to passage your cells in 10% FBS only. Nevertheless, you can try to grow them in 20% FBS also. For that, you may have to adapt your cells accordingly. For more insights, I recommend you to read this interesting research article.
Addressing the impact of different fetal bovine serum percentages on mesenchymal stem cells biological performance. DOI: 10.1007/s11033-019-04898-1.
Good luck :)
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I need to transduce HL-1 cells and keep them for 5-7 days for the transgene to be expressed but they proliferate very fast, get over-confluent and die around day 3. Etoposide was very toxic for this cell line and decreasing serum concentration did not help. Any idea how I can stop/slow down the cell proliferation?
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You can seed HL-1 cells with medium serum-free. But I advice you to assess if this protocol change will influence your experiment. In all case, you shloud consider the option given by Dr. Zhou
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I am starting to investigate cell migration (A549 cell line; wound healing assay) for longer than 24 hrs. I want to make sure I am looking only on migrating but not proliferating cells.
Are there any suggestions which proliferation inhibitor I can use, which otherwise don`t inhibit cell migration?
Thanks
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A low concentration of mitomycin C may suit your experiment - here is a paper detailing the experiment in A549 cells:
Alternatively, you could use thymidine to block DNA synthesis at the G1/S phase boundary
Good luck with your experiments.
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Are there mechanics by which a cell can recognize how many cell divisions it has underwent therefore regulating certain pathways? Or are there indicators which scientists can observe to determine how many cell divisions have occurred after a drug treatment?
Of course you can use the FUCCI system and FACS to determine the particular cell stage a cell is in. You could also theoretically count cells to determine how many times the cells in general have divided inferring cell divisions.
Thank you,
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In a normal cellular process, a small portion of telomeric DNA is lost with each cell division. When telomere length reaches a critical limit, the cell undergoes senescence and/or apoptosis. Telomere length may therefore serve as a biological clock to determine the lifespan of a cell and that of an organism.
Measurement techniques that provide the distribution of telomere lengths in a cell population offer improved and more informative data regarding telomere length dynamics, and one such technique is Telomere length Combing Assay (TCA) which is an accurate and robust technique to measure the distribution of telomere lengths in a cell population.
Please refer to the recent article below for more information.
I hope this information helps you.
Regards.
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I was asking myself why we usually change the media just the day after thawing the cells. It seems that the two reasons most of the people exposed are:
1. To get rid of the DMSO leftovers.
2. To remove the dead cells floating in the media as soon as possible.
Without entering to discuss whether the small amount of DMSO in our media could be affecting cell proliferation or not, I was wondering about the necessity of getting rid of the dead cells. Apoptotic cells are known to release factors that increase wound healing and cell proliferation both in vivo and in vitro.
Do we have any research that supports that these dead cells could not be beneficial for the first days of culture after thawing?
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Everybody is bringing up valid points concerning the possible negative effects of dead cells on the remaining live ones. However, I am missing hard data. Does anybody know some? Everything I read here is intelligent guesses based on (valid) theoretical considerations and anecdotal evidence. Unless there is reliable data, I would not declare this issue as settled. And there are many different cell types out there, which might behave also different when it comes to dead neighbors...
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What additives can I add to the culture medium to improve the expansion of a Jurkat cell in a 96-wells plate?
I have tried to isolate a transduced single jukat cell without a selection marker.Then I expand it to a large number of jurkat cells. but during culture, jurkats become apoptotic and die after 3 weeks. why?
what can I do to have a clonal jurkat cell line from a single cell?
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The methods in the article I linked above describe how the authors performed limiting dilution of human T cells.
The formulation of standard RPMI 1640 does not contain pyruvate, but it does contain all amino acids, so cells leaking small amino acids should not be an issue.
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Is that CCK-8 test can also react with DH5a (E.coli)?
In numerous studies have shown that CCK-8 or MTT tests for detecting the proliferation of cancer cells incubated with certain bacteria. However, in my recent work, I find that DH5a also presents a great influence on the OD450 of the CCK-8 test. I really wonder the validity that using this method for examining the proliferation of co-culture bacteria and cancer cells. Furthermore, I apply Hematocyte Counter for checking this result, I find that the so-called phenomenon of cancer cells proliferation have been definitely disappeared, or even displayed cells number decreasing. I did not know how to explain these conflicting results.
Many thanks for your kindly response.
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Kaili Fu I think it difficult to observe cell proliferation. Actually, I tend to consider that the coculture of bacteria and cells in vitro may be a competitive relationship. The CCK-8 assay, Edu kit, and Flow Cytometer always present the confusing result, because the live bacteria can also react to these reagents. I did not try the LDH assay, but many species of bacteria also carry the LDH enzyme.
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My substance decreases percentage of cell numbers in the G1 and increases percentage of cell numbers in the G2 phase while percentage of cell numbers in the S phase(cell cycle) remains same compared to the saline control in the cytometry assay. WB shows unregulated expression of Cycline D1.What can this suggest? (Btw, CCK-8 assay show higher proliferation rate in treated cells)
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Hello Muhammad, What cell numbers are you recovering from the different treatment groups? And how are you evaluating cell cycle eg DAPI, Ho, PI ? What about % apoptototic population in the different treatments is that changing between groups?
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Hello
Does anybody know which would be a good assay to assess cell proliferation in transwells?
I want to understand if my treated cells proliferate at a higher rate compared to the unstimulated ones. I thought of using BrdU but I don't know if is going to affect the cells (toxicity) for a longer time period than 16 hours.
Thanks
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Antonio Greco , the EdU kits available now are simpler than doing a BrdU assay, but it should be possible to visualize both with microscopy . Adity Pore's suggestion of using CellTracker dyes should also work for fluorescent microscopy, and we use these same dyes to track cells during chemotaxis assays (i.e. transwell migration/invasion).
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I induced ACh and SFb with TNFa and INFg for 96 hour. On the cell viability assay, I saw an increase in number of the cells in comparison with control condition. I read papers reporting that cell proliferation should be inhibited. Thus, reducing cell number.  Any explanation for this?
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There is an explanation. ACh can both increase and inhibit cell proliferation depending on the concentration.
See my publications and monographs.
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I am looking to incrementally increase calcium ions in culture media to assess the impact on cell proliferation. I was hoping to using CaCl2 anhydrous prills. Can someone advise on how to acheive this? Ie dissolve CaCl2 in dH2O and add to media or dissolve directly into Ca-free DMEM?
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Fabricio Amador López thank you very much! Very helpful advice and tips
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I want to mark parasites with CellTrace ™ Violet Cell Proliferation Kit, for flow cytometry (Catalog number: C34571). Has anyone used it with parasites? I work with T cruzi.
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Dear Sofia!
Please you look at the article:
2.2. CellTrace Violet in vitro assay Trypanosoma cruzi trypomastigotes were isolated by centrifugation and allowed to recover for 2 h at 37°C in high-glucose DMEM medium with 10% FBS, and then labelled with CTV fluorescent dye (Thermo Fisher Scientific), according to the manufacturer’s protocol. Briefly, 2 × 106 trypomastigotes were washed in PBS and then incubated for 20 min at 37°C in 10, 5, 2 or 1 µM CTV, protected from light. Unbound dye was quenched by the addition of one volume FBS and incubating for 5 min at 37°C. After washing (×2) in fresh complete medium, trypomastigotes were used for infection. Vero cells maintained in RPMI 10% FBS were trypsinized and seeded at 104 or 105 cells per well in 24-well plates containing coverslips, or in eight-well Ibidi µ-slides, and allowed to attach for 6 h before infection. Trypomastigotes were added at a multiplicity of infection (MOI) of 10 : 1 (parasite : host cell) and allowed to invade overnight (16–18 h). Cultures were then washed with PBS (×3) to remove non-invading parasites, and infected cultures incubated in RPMI with 2% FCS. Coverslips were fixed at different timepoints by transfer into a plate containing 4% paraformaldehyde for 30 min, then stained and mounted for microscopy using Vectashield® with DAPI, or with propidium iodide following RNase treatment.
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Dear Fellow Researchers,
I'm having some troubles with BrdU assay. I provide you my simplified protocol and images in attachments below. I am not able to count proliferating cells beacuse of these blurry green shapes that do not locate in the nucleus. It worked several times with PC3 cell line but from some time I'm having troubles with other cell lines (BXPC3, HCT, HT-29). Could you please tell me what should I check?
Protocol:
1. Add BrdU stock (4ul/l) to the medium for a given time (usually 24h). Final concentraton 10 uM.
Next day
1. Remove the cell culture medium.
2. Wash the cells with warm PBS.
3. Fix the cells in 70% cold ethanol (keep in room temperature for 20 min).
3. Wash the cells witt PBS (5min, RT)
4. Wash the cells with PBS + 0.5% triton X100 (2x5min, RT)
5. Incubate with 0,5 ml of PBS and 0,5 ml of 4N HCl - incubate 30 min in RT
6. Wash the cells twice with PBS (2 x 5min)
7.Incubate in 1 ml sodium borate 0.1M (1min)
8. Wash the cells twice with PBS.
9. Incubate with 1st ani-BrdU (mouse) 10ul/ml in PBS+1%BSA+ 0.5% tween (1h, RT, dark)
10. Wash the cells with PBS+0,5% tween twice (2 x 5 min)
11. Incubate with 2nd anti-mouse 1:650 in PBS+1%BSA+0,5%tween (1h)
12. Wash cell with 2 x PBS+0,5% tween (2x 5min)
13. Incubate with DAPI (15 min)
14. Wash the cells with 1 x PBS (RT, 5min)
15. Mount coverslips with fluoromount G.
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Dear Mateusz Kciuk many thanks for your interesting technical question. As an inorganic chemist I'm far from being a proven expert in this field of research. However, I can suggest to you some potentially helpful literature. For example, please have a look at the following relevant link:
Top Tips for Troubleshooting Your BrdU Blues
It might also be helpful to have a look at the answers given to this closely related RG question:
Has anyone experienced problems with BrdU staining the cytoplasm instead of the nucleus?
(9 answers)
I hope this helps. Good luck wth your work! 👍
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I'm working with HEK293 cells and I'm trying to find a way to increase the expression after virus transduction. I'm thinking about a way of inhibiting cell proliferation and direct the cell more into transcription and translation. Do you think there is a chemical to do that ? I know that Thymidine block can inhibit cell proliferation. However, I 'm more interested in increasing the expression.
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Hi John, Thanks for your answer. I am using a maximum concentration of the virus. From my experience, there is a limit of increasing the expression after a specific concentration. Tat is why I think I have alredy reached the limit of the amount of virus used.
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I transfected px458 in Expi293F HEK cells with lipofectamine and then sorted single cell by FACS into 96 well plates.
The medium is Expression medium without antibiotics.
The cells are cultured in a 37C, 8% CO2 incubator with shaking.
However, after one month, no cell proliferation has been observed.
If anyone has done transfection with Expi293F and can tell me the solution or protocol, I would appreciate it.
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I didn't work on HEK, but i tried to sort single K562 cell into 96 well plates, and recovered 84.5% positive clones.
To me, the main stress for these HEK cells come from transduction reagents. After transfection, wait a few days for the cells to calm down and start to grow, and then sort cells into 96 well plate may be helpful.
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I found in the literature that nicotine, acting as an nAChR agonist, increases the proliferation of A549 cells (human lung adenocarcinoma cells). But when I treat A549 cells in 96-well plates with nicotine (concentration range from 0.1-5 uM) for different periods (from 1 to 3 days), I get no effect on cell proliferation (using alamarBlue assay, Neutral Red Uptake assay and microscopic observation). For experiments I am using DMEM (high glucose) cell medium supplemented with 10% FBS. Does anyone have an explanation as to why I am not getting an increase in cell proliferation?
I would like to thank you in advance for any answers/comments.
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Dear Veno!
You do not observe cell proliferation with nicotine because you are using inappropriate assessment methods.
For example,
Proliferation Assays
Bromodeoxyuridine (BrdU) labeling kits were obtained from Roche Biochemicals. Cells were plated in poly-D-lysine coated chamber slides at a density of 10,000 cells per well and rendered quiescent by serum starvation for 36 hours. Cells were then re-stimulated with 1µM nicotine or 10% FBS for 18 hours. S-phase cells were visualized by microscopy and quantitated by counting 3 fields of 100 cells in quadruplicate. Data is presented as the percentage of BrdU positive cells out of the 100 cells counted.
Figure 1
Nicotine can promote proliferation, invasion and survival pathways in A549 NSCLC cells. (A) Nicotine can induce S-phase entry in A549 NSCLC cells in a dose-dependent manner, as measured by BrdU assays. (B) Nicotine can induce anchorage-independent growth of A549 cells in soft-agar assay. Nicotine increased the size of the individual colonies but had little effect on the total number of colonies. (C) Nicotine can promote migration and invasion of A549 cells in Boyden Chamber assays, in a dose dependent manner, the maximal effect being observed at 1µM nicotine. (D) Nicotine can confer resistance to anoikis of SAECs. SAECs were treated with 1µM nicotine and plated on polyhema coated slides. Apoptosis was measured by TUNEL assays. (E) Treatment of A549 cells with 1µM nicotine caused morphological changes, similar to VEGF on 3D collagen gel assays.
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I have cultured the MCF7 and T47D cells, after 4 times of passage the cell proliferation was slower than before and the cells detached. After the cells washed by PBS, they still detach the next day. Then, I checked the pH of DMEM+10% FBS. It showed 8.25.
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Hello Rohmi
The requirement of CO2 in cell culture is important because it dissolves in cell culture medium where a small proportion of it reacts with water to form carbonic acid which in turn interacts with the dissolved bicarbonate ions in the medium to control a stable physiological pH through the bicarbonate buffering system. The amount of sodium bicarbonate in the medium will decide upon the amount of CO2 to be used to maintain the pH. The physiological pH is generally in the range of 7.2 to 7.4.
DMEM medium has a higher concentration of sodium bicarbonate (44mM). So it will require a higher percent of CO2 8-10% compared to other cell culture media. Though many still use 5%CO2 for DMEM as information on this aspect is still scanty. Using DMEM at 5% CO2 will result in a pH of 7.5 which although acceptable for most cell cultures is slightly outside the desired range of physiological pH.
I would suggest you change your culture medium from DMEM to EMEM as EMEM is formulated with a much lesser concentration of sodium bicarbonate (26mM) supplemented with 10%FBS.
The pH of 8.25 does not seem to go well with the cells.
All the best.
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Hi all,
is there a way to analyze cell proliferation with CFSE with BD DIVA software? I activate murine splenocytes or purified CD4 and I use Cell Trace and follow the exact protocol but I only see the initial peak on Day 0 as expected. I harvest the cells each day from day 1 to 5 but instead of seeing multiple peaks, I rather see a "curve" each day, almost falling at the same place of intensity. Any ideas?? Should I only harevst the cells on day 4 or 5 (final)?
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Hi Spyridoula,
You can try titrating down the CFSE to optimize the peaks. You should also gate on CD4+ live cells. Other advice I have is to:
1) look at D0 AND d4 or d5 only, not d1,2,3
2) change your collection size. For example, for D0 (or unstimulated), collect 5k CD4+ events but for d4 (or 5) collect 50-80K events so that the maxima of the 2 curves are about the same height. If you look closely, you will notice that there are humps starting to appear, you will see them resolve better with titrating down the cfse and by collecting many more events
Kind regards,
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I want to conduct a proliferation assay to test a chemotherapy combination on a cancer cell line. At first, I thought about the MTT assay. But now, all sources are saying that these do not measure proliferation.
If you had a hypothesis that some drugs decrease proliferation, how would you go on about it? what experiments/assays would you use?
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Hello, Aicha!
1. Probably the simplest analysis for proliferation is to sow the same number of cells, treat with drugs and after the same time count the number of cells (for example, in a Goryaev chamber or using an automatic cell counter). Then, in relation to the ratio of the grown cells to the initial number, the proliferation index in% can be estimated.
2. You can use antibodies to marers of proliferation Ki-67, PCNA and use flow cytometry or immunofluorescence.
3. MTT test can also be used. The viability of cancer cells will be proportional to their proliferation. If your drugs act on proliferation, then cell viability will also change.
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Need to detect cytokines from PBMCs dosed with peptides. This is to determine if the peptides elicit PBMCs to release markers for immune response. I am unsure how long to incubate the PBMCs with the peptides? And.. Do you need to dose the cells multiple times?
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Hello!
You can look at this protocol:
4.4. Ex-Vivo Induction of Cytokines from rhIL-1β and LPS-Stimulated PBM
The PBMC were placed in 24-well plates after the density adjustment at 1 × 106 cells/mL (final volume of 1000 µL). Cells were cultured in RPMI-1640 medium without serum and supplemented with antibiotic and antifungal. Untreated cells were taken as the control group for each experiment. The effect of LPS (30 ng/mL) in the secretion of TNF-α was studied in samples from four HBD to demonstrate the positive stimulation of the cells
LPS response in PBMC was determined at 0, 10, 30 and 50 µM. Subsequently, PBMC culture was performed with LPS (30 ng/mL). Each experiment was done with two biological replicates and was incubated for 24 h at 37 ◦C in a humidified 5% CO2 atmosphere.
4.5. Cytokine Analysis by Multiple Analyte Profiling (xMAP) Technology The concentration of IL-1β, TNF-α, IFN-γ, IL-6, IL-12, IL-17A, M-CSF, GM-CSF, IL-2, VEGF, IL-15, IL-7, IL-1Ra, IL-4, IL-5, IL-13, IL-10 and IL-9 and were quantified from culture supernatants by multiplex immunoassay method using the Bio-Plex ProTM Human Cytokine 27-Plex Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Luminex MAGPIX® (Luminex Corporation, Austin, TX, USA) instrument was used for xMAP assays
Or
2.3. Whole Blood Stimulation Heparinized whole blood was taken from healthy rats and added in 96-well plates (200 μL/well), supplemented with different concentrations of each stimulating reagent (PMA: 1, 5, and 25 ng/mL; Ionomycin: 1 μg/mL; PHA, LPS, Con A, PWM: 1, 5, and 10 μg/mL), and incubated for 0, 2, 4, 6, 8 or 10 h at 37 °C with 5% CO2.
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Hello,
I want to measure the proliferation of magnetically isolated T cells. I have successfully stimulated PBMC with 5 ug/mL PHA-L and measured their proliferation with MTT. The same method has not been successful in my magnetically isolated T cells (>95% CD3+).
I would like to know if PHA-L alone will stimulate the proliferation of isolated T cells (>95% CD3+) or if an additional stimulant is required?
Thank you,
Thomas
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Dear Thomas Byrne,
Yes, it is possible to stimulate the magnetically isolated T cells. You may consider using interleukin-2 (IL-2), a potent T-cell mitogenic cytokine along with PHA.
Hope this helps.
Best - Pradyumna
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Once I have carried out the research I have to insert not one but more journal articles, books and more (more preprints, more groups of purposes and more groups of hypotheses) as a set of results of research. So, I would need multiple cells to enter the various articles, various books, multiple purpose groups, and multiple hypothesis groups that the research has implemented. You tell me very simply how to do it?
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I think that it depends on many factors including: where you want to publish? Which journal, the amount and quality of your data, the available funding fees for publication, the philosophy and approach of your work, type of publication( book, preprint, review, articles ) ????. For example, you can decide to publish it one by one cell if you have enough pushisable data per cell lines. Otherwise you can groups the results with many cells lines and publish it.
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Hello
I am doing a study using Crispr/cas9 edited cell where we KO a gene and study the phenotypes associated with it.
However, our data showing opposite phenotypes (i.e in the first study we observed decreases in cell proliferation, migration and cell growth, and in other study using same cell model we observed increases in cell proliferation, migration and cell growth !). We had repeated the experiments so we are confident that we have true data. I am looking for a scientific explanation.
Does the mutation created by Crispr cas9 system lead to somatic mutation by which tumour cells change their behaviours over time ( complete acute vs complete chronic loss )?
Or is it possible that Crispr cas9 system for a specific gene creates driver mutations ‘and then passenger mutations or vs?
Thanks in advance.
Bayan
Note :
“Driver mutations confer a growth advantage on the cells carrying them and have been positively selected during the evolution of cancer”
“Passenger mutations hat do not confer growth advantage, but happened to be present in an ancestor of the cancer cell when it acquired one of its drivers”
Refr. Stratton, Michael R et al. “The cancer genome.” Nature vol. 458,7239 (2009): 719-24. doi:10.1038/nature07943
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Interesting question. Following the discussion.
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Hello everyone!
I have the following question: I tried to expand CD4 Tregs in vitro to get enough material for WB or any other application. This is my first trial with them.
So, I`ve got FoxP3+ CD4 T-cells from mice (I use FoxP3-GFP mice) by FACS and activated them in vitro for 3 days with DynaBeads as beads/T-cells 1/2 ratio with rhIL2 ~100 U/ml. By that time the cells were mostly alive, looking good and being >65% GFP+. I`ve removed beads from the culture and rested the cells for another 6 days on rhIL2 100 U/ml dividing them every 2-3 days.
When I checked them, only ~23% were GFP+ and they didn`t secrete any TGFb or IL10 upon PMA/Ionomycin stimulation, but expressed quite high level of IL2 and TNFa (even GFP+).
I`ve checked protocols, but mostly all of them say that I just need to sort cells and activate them and then culture with IL2 100-1000 U/ml (quite a huge variation range).
What did I do wrong?
I`m quite fresh with this expansion method, so I`d be very grateful for suggestions.
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Hi Dmytro,
The different protocols for Treg expansion include a very broad range in IL-2. You are at the upper limit of IL-2 concentration and you are correct to assume that if you use higher IL-2 levels, you are most likely going to observe expansion of other T cell populations (in the context of bead activation). That is the basis of CD25 /T reg function.
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Hi,
I'm currently working with the Flp-In T-REx system to get isogenic stably transfected cells expressing my GOI (gene of interest, of about 1.4 Kb).
I have generated a Flp-In Cell Line and since I'm not interested in modulating gene expression via Tetraciclin, I decided to direct cotransfect my clone with the pCDNA5/FRT/TO_GOI and pOG44. As suggested by the user manual, I independently repeat the experiment using the pCDNA5/FRT/TO_EV (empty vector). After 24 hours hygromycin was added in all plates. After about a week I started to get clones in both the plates and now I'm propagating them.
Here comes the point.
There is clear evidence of larger and faster-growing clones in the cells transfected with the pCDNA5/FRT/TO_EV compared to those transformed with the pCDNA5/FRT/TO_GOI. Since the lack of positional effect is one of the features that characterize this system I can't understand why this happens. Having in mind to test how the expression of my GOI in both stimulated and unstimulated conditions may influence several parameters (including cell proliferation and viability) this makes things more complicated.
It could be possible that my GOI has some effect on cell proliferation, but since it is a receptor that needs a stimulus to be activated, I don't think this is the reason why this occurs.
Any explanation or further suggestions for testing alternative hypotheses?
Thanks in advance.
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Hi,
Just reading the manual and it looks like transfecting pcDNA5/FRT/TO EV shouldn't induce Hygromycin resistance. I am using the Flp-In T-REx system and am observing Hygro resistant foci in flasks of cells transfected with pcDNA5/FRT/TO + GOI and also in pcDNA5/FRT/TO EV. I don't think this should be happening!
VG
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I want to detect that my proliferating cells underwent a DNA Damage after treatment with drugs. I already know the H2AX method but looking for other methods. Many thanks in advance!
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I hope the paper attached will help you more.
Tunnel assay, comet assay, H2AX phosphorylation, immunocytochemistry and few other methods are mentioned in these.
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Background info:
I have calculated the doubling times of wild-type cell lines and gene knockdown cell lines. Growth curves were measured three times (day 0-day 6), each time there were 2 technical replicates. The technical replicates were plotted over time and via log-linear regression a doubling was derived.
I now want test whether knockdown of this gene affects doubling time. As the variation between the different growth curves (doubling times) is quite large (likely due to random things like people opening the incubator more frequently that week and differences in confluency at plating, things that are the same for both wild-type and knockdown cell line), I think I need to use a paired-t test.
However, from what I've seen, a paired t-test does not take into account standard error of those doubling times. So I'm wondering, is this correct? I do not have a background in statistics, but this feels somewhat wrong.
To clarify: for both the wild-type cell line and for the knockdown cell line I have three doubling times. I want to compare these to see if the knockdown has an effect on doubling times. As I derived the doubling times from log linear regression I think it's best to compare the slopes rather than convert those slopes to doubling times and compare those.
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I'm not sure where you found that t tests do not take into account standard errors of means. T-tests are based on the t statistic, which is the quotient of the mean difference and the standard error of the difference.
Do you mean that the t-test only take into account variability between growth curves of individual cells, but not the variability of each curve? T-tests assume that individual data point (in your case growth curve slopes) comes from a single measurement. One underlying assumption of t-tests is that measurement error is distributed normally, so the average measurement error will approach (or be) zero as the number of data point increases.
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I have been differentiating multiple iPSC lines into NPCs and then into astrocytes. I would like to do some qPCR to further characterise my iPSCs and astrocytes but was wondering how best to choose a housekeeping gene. I've tried 3 different ones for all cell types from all lines (GAPDH, YWHAZ and b-actin). Should I try and find the least variability across cell type, or across cell lines? Or is it okay to use different housekeeping genes for different cell types?
Sorry if I've explained this very badly! I'd appreciate any advice. :)
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I recommend you the database "Housekeeping and Reference Transcript Atlas" (www.housekeeping.unicamp.br). It offers candidate reference transcripts stable across multiple tissue types.
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I am culturing a spheroid which derived from intestinal stem cells. The colony seemed to contain multiple types of cells and I would like to identify what kind of cells in the colony.
Does anyone know how to identify the cell types in heterogeneous cell cluster?
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Dear Dr. Saleh Alkarim
Thank you very much for your reply.
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Mean pore size of tissue engineered scaffold plays a vital role in cell activity(eg. Infiltration of cells into the scaffold, proliferation etc)which in turn is dependent on cell type. In order to optimise electrospinning parameters to produce a viable scaffold for culturing human embryonic stem cells, the mean pore size would serve as a optimisation target to predict cell activity.
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The following article may be useful.
All the best
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Hello,
I am examining the phosphorylation of the MAP kinase ERK by immunofluorescence. In order to do this, I understand that serum-deprivation of cells will eliminate background phosphorylation levels. For how long should I serum-deprive the cells? Shall I keep them in low serum for a full passage prior to seeding in my assay plate? Or shall I seed them in the assay plate in low serum? Or shall I seed them in the assay plate in normal serum and then change to low serum after cell landing? How many hours of serum-deprivation will result in low background phosphorylation? I imagine that 24 hours at 1% serum should do the trick. Alternatively, should I eliminate serum altogether? After how long in low-serum or serum-free conditions will cells signal abnormally or arrest?
Thank you!
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You can culture the cells in serum-free medium for 3 days and keep cells that are not deprived of serum as negative control for examining the phosphorylation of the MAP kinase ERK by immunofluorescence. For more detail kindly see the published paper in FEBS Lett. 2008 Aug 6;582(18):2703-8.
doi: 10.1016/j.febslet.2008.06.051. Epub 2008 Jul 9.
Serum starvation induces H2AX phosphorylation to regulate apoptosis via p38 MAPK pathway.
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Dear all,
I used the Click-it EdU flow cytometry assay to measure cell proliferation of my samples. However I do not know how to analyse them and interpret. How can I use the data to compare cell proliferation between two different groups?
Thanks :)
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Dear Sofia at the first, gate the live cells. next, use histogram to discriminate unstained cells, stained but not stimulated cells, Positive control (which stimulated by PHA, Antibody or Ca/ionomycin ...) and your interested cells. finally compare the percentage of proliferated cell in the interested groups by proper statistic.
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hi everybody,
i have some troubles with chemokines and cell proliferation. Different papers evidence that CXCL8 (but also CXCL12, CCL2 and other chemokines) are able to promote cancer cell proliferation. Based on these statements i tried to assess if CXCL8 would increase TPC-1 and 8505C thyroid cancer cell proliferation in vitro. Firstly i seeded my cells in a 96 well flat at a density of 4000 cells x well. After adhesion i treated cells with increasing concentrations of CXCL8 (10, 50 and 100ng) for 24 48 and 72h . At the end of each time of treatment i stained cells with cristal violet 0.5% fixed with METOH. I read the OD and calculated my results basing on control untreated. However my results were not reproducible. I tried also to assess the proliferation by CFSE at FACS but i did not found any increase of cell proliferation after treatment with CXCL8. I do not understand what's wrong, i have probably choose not adequate proliferation assays? could anyone suggest me some alternatives?
Thankyou
Francesca
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Francesca Coperchini Yes, but you can just add it in your wells and visualize in brightfield. WHile it's cheap and fast, it can undermine your results though, but you can give it a go for a first kind of interpretation. Add a control on 0 hour timepoint as well
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I want to check the effect of IL2 on the phenotype of a population of mouse CD8 T-cells I'm studying. So I want to stimulate my cells with IL2, anti-CD3 or anti-CD3+IL2. Recently I've ordered 100 mcg human recombinant IL2 from Peprotech.
In the manual it's suggested to dissolve powder in 100 mM sterile acetic acid as 1 mg/ml. I equilibrated temperature of the vial, centrifuged, dissolved in 0.1 M acetic acid, waited for 3-5 minutes to give better solubilization. Then I dissolved my protein solution additionally to 50 mcg/ml in sterile PBS/0.1% BSA, aliquoted and froze at -80. 1 vial was diluted more in order to get 20000 U/ml as the instruction claimed IL2 activity to be 10 millions U/mg. So as I got 100 mcg IL2 it means I got 1 million units IL2.
But this IL2 added to get 50 U/ml doesn't support CD8 T-cell survival neither alone nor during polyclonal T-cell stimulation and even decrease their survival what is strange.
Why it is so? Any ideas? Any suggestions?
Many thanks!
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Cell stimulation protocol is like that:
Sorted CD8 T-cells are either cultured at IL2 50 U/ml or at plate-bound anti-CD3+soluble anti-CD28 or at plate-bound anti-CD3+soluble anti-CD28+IL2 50 U/ml.
As I tried before IL2 induced my CD8 T-cells to proliferate even at 25 U/ml.
Now it is not only supportive alone but it even reduces anti-CD3 induced proliferation.
Any idea where the mistake can be?
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Have anyone tried to transfect a cell multiple times with same DNA. Like a cell line which is already made stable with a particular DNA can be transfected again with the same DNA which is already integrated into the genome?
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Beera Shankar Anand Most likely you will not see the increase in expression of transgene, or at least not to the level you want to see. The expression level of transgene depends more on the kind of promoter you use than on anything else. If your plasmid has a weak promoter, try redesigning the plasmid.
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Dear Scientists,
I am working with a gene of interest in colorectal cancer, which has no role in cell proliferation. In addition, by knockdown this gene, the FAK and SRC protein expression are decreased. I have changed amino acid by SDM and check the protein expression of the gen by WB, the expression is decreased and the MW was less than actual MW. Then, I check the cell proliferation after transfection with mutated plasmid and wild type plasmid, I have noticed a significant increase in proliferation of the mutated one compare to WT. Can anyone have an idea to explain how this is happening?
Thank you in advance,
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Hi Colin,
Can you please explain what do you mean by ''Receptor and expression factor dilution upon division?''?
Thank you,
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In my experiment I am currently growing two strains of bacteria, and in order to test them in my experiment I have to grow the biofilms in 15 mL falcon tubes. I haven't yet found a great assay to determine cell proliferation. I have been running CV staining of biofilms then de-staining cells, and transfering that solution to plates to collect abs. I am open to any suggestions.
I was thinking about getting a OD 600 on the media that I pull off the tubes after 24 hr incubation, but was concerned that I wouldn't be able to quantify the biofilm growth. Thank you for any help you can give.
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Thank you for both of these papers they helped clarify the problem with using plastics. I really appreciate your help and knowledge! Thanks again! Mert Sudagidan
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Stem cells can reproduce themselve, although their proliferation rate is very low.
On the other hand there are the so-called "immortalized cell lines" which have a normal or high proliferation rate, and can sustain it over long periods of time.
Can stem cells also be considered to be a kind of such "immortal" cells?
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Vad Pérez Stem cells are generally divided into two (very broad) groups, pluripotent stem cells and all other stem cells (such as neural progenitors, muscle progenitors and other progenitor cell types). Pluripotent stem cells can be either embryonic stem cells obtained from embryos ("ES cells") or induced ("iPS cells).
The growth rate of stem cells varies widely depending on the cell type. The same thing can be said about cancer cells. For example, I worked with more than 20 different cancer lines, of which about 8 were neuroblastoma cell lines. Neuroblastoma in particular is very hard to grow, has extremely high cell death rate, is prone to differentiating, and many lines actually grow slowly. Not all cancer cell lines grow rapidly. However, it is true that all of them are immortal (otherwise it would not be called a cell line).
Stem cells, in turn, are very different depending on the cell line, too. For example, now I am working with two human embryonic stem cell lines, KhES-1 and KhES-3. They have very different growth rate, and one grows much better than the other. To illustrate, my KhES-1 is still "passage 4", while my KhES-3 line is already "passage 10", even though I started them and split them always at the same time (because I work on both at the same time). The speed of their growth is quite different. ES and iPS cells can definitely divide just as fast as HeLa or faster. The speed of division does not directly correlate with immortality.
Now, about immortality, yes, all pluripotent stem cells (that is, ES cells and iPS cells) can divide indefinitely. However, whether the mechanism of this immortality is same as in cancer, is not clear. Pluripotent stem cells do activate telomerase, and extension of telomeres happens during reprogramming. They are very similar to cancer cells. The question "what is different between cancer cells and pluripotent stem cells" (i.e. why cancer is so aggressive while pluripotent cells are not, even though both are immortal) has not been answered yet. Whoever answers it, is going to publish pretty high.
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Hi all,
I am running a n experiment involving primary rat Schwann cells ( passage 3) and varying dosages of H202 (0mM, 0.1mM, 0.3mM, 0.8mM, 1mM, and 2mM). Schwann cells ( n=2,000) were grown on a 96 well plate for 48 hours in 10% DMEM media with glutamax and forskulin. The varying concentration of H202 ( previously mentioned) was added to media after 48 hours and cells were subsequently grown in 1%FBS media. CellsThe experiment was done in triplicates. The results of my experiment seemed to show an increase in Schwann cell proliferation after CCK-8 assay. Is this to be expected or is it a technical error?
Please see attached for my data results
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It makes sense to me too. Does this protein reacts with NADPH too?
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I prepared TiO2 suspension in DMEM medium and used it to seed plate. Under the microscope I can observe something is moving and vibrating. Does the particles move and vibrate or it is cause bacteria contamination? Does anyone encountered similar problem as mine? Besides, the absorbance reading of the control and treated cells are almost same or sometimes more than control but the cells are still alive. Does it caused by contamination or the cells proliferate when the reading is higher than control? I need some advise on this.
Thank you in advance.
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Hi Pravena, TiO2 should be insoluble in DMEM, so you have a suspension, but depending on the size of TiO2 particles you can see something under the microscope or not. If your TiO2 particles are big enough, or being them nanoparticles, they aggregate forming bigger ones, then you can see them wanderig and vibrating due to the Brownian motion
If your particles are nanoparticles, and they do not aggregate, you cannot see them with an optical microscope, therefore, what is moving in your sample should be some kind of cell... may be bacteria contamination.
I hope it helps. Best!.
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If we have a plate of stem cells consist of one million cells in the start of in vitro differentiation process, after the induction of differentiation factors, and after the end of differentiation,
How many cells usually remain? (one million?)
What happens for cell cycle and cell division process? These process will be stop during differentiation?
Is there any publication about the genetics ans cellular characteristics of the cells after in vitro differentiation?
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There are lot papers which described about cell cycle activity during differentiation. You are right that cell proliferation need to be stop, so cell can go for differentiation.
How any differentiated cells expresses a particular markers which undifferentiated cells do not expresses. Similarly, for proliferation and apoptosis phenomenon. Apoptosis assays will determine whether differentiation induction caused any death or not.
While, to track the cell proliferation, you can use FLOW CYTOMETRY analysis for Ki67, which is prominent proliferation marker, also PCNA & MCM2 expression. Even several dyes are available such as BrdU, EdU can be used.
Ki67 expression before and after differentiation will give very good answer whether cell exit the cell cycle or not...
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Hi! I would like to compare cell proliferation rates.
The working hypothesis is that the proliferative effect of extracellular vesicles on cells cultured on the skin implant is increased compared to samples with pure cell culture.
There will be 4 samples:
1) cells (control)
2) cells + skin implant
3) cells + skin implant + extracellular vesicles
4) cells + extracellular vesicles
Cells will be from 3 donors, and experiments will be carried out 3 times with each donor culture.
Can someone help? Could you advice what method is the best?
Thank you in advance, your help is much appreciated.
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In R the model could be formulates like
model = glmer(rates ~ implants*vesicles + (1|donor), family=Gamma)
where rates are the rate constants detemined, implants and vesicles are binary factors (yes/no) and donor is a factor identifying the subject (used a random intercept factor in the model).
You are interested in the interaction of implants and vesicles. The difference-in-difference statistic can be obtained with
summary(model)
and you can get a p-value with
anova(model, test="Chisq") or drop1(model, test="chisq")
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Hello everyone! I regularly use the Sigma BrdU cell proliferation kit for my cell assays, and due to the nature of the kit I always run out of some reagents before others. This means I keep repurchasing the same kit just to replace one or two components since they don't sell them separately, and so I have accumulated multiple vials of the BrdU labeling solution as well as the wash buffer. It makes no sense to keep buying this kit since it is quite expensive. I would like to know if anyone has any experience in replacing the fix-denaturing solution, the anti-BrdU-peroxidase antibody, the antibody dilution solution, or the substrate solution, as a DIY reagent and still have the assay work just as well????
As a side note, I now use MTT instead of BrdU for cell viability because its much cheaper, however I am repeating a particular set of experiments from last year where I originally used the BrdU kit, so I would like to use the same method for consistency purposes here so I don't have to re-do everything with MTT....
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One thing I tend to run out of quite quickly from that kit is the stop solution, and I've found that using either H2SO4 or HCl at standard ELISA concentrations works just fine, so that I can make the kit last a bit longer. Good luck!
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We wish to use the Click-iT EdU Plus Kits to visualise cell proliferation of mouse splenocytes ex vivo.
One method of detecting and visualising EdU incorporation is fluorescence IHC. However, the protocol recommends paraffin-embedding the tissue before staining. Our problem arrises because we would like to multiplex EdU staining with our antibody of interest, which works much better in OCT-cryopreserved tissue than in paraffin-embedded tissue.
Has anyone used the EdU kits and subsequently frozen the tissue for fluorescent IHC?
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I have not personally tried to stain samples with incorporated EdU, however, I have EdU after being frozen at -20 C for years. I do not see any reason why freezing a sample with incorporated EdU wouldn't work, as long as you are performing the EdU staining/detection after you pull the frozen samples out, presumably when you are doing your antibody staining (or immediately before if using PE/PE-conjugated Abs)
Good luck!
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Do hypoxic or necrotic cells release chemicals that inhibate the proliferation of the surroundings cells ?
And do you think that such chemicals could alone stop a multicellular spheroid from growing ?
Meaning I could understand a spheroid stops growing because that there are not anymore anough nutrients in the media, or because there is a flow of proliferating cells towards the center do to the fact that cells are dying there. But could the inhibition due to chemicals be a cause of the cells stopping to grow ?
(I'm asking that cause of some experiments I'm observing)
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Hi Joesph,
I don’t think I’ve come across any literature that suggests that hypoxic cells will inhibit the growth of another non-hypoxic cell. I do, however, believe that cells in the interior of the spheroid (i.e., quiescent and hypoxic) know that the nutrient supply is low. Hence, quiescent cells undergoing growth arrest to ensure that they survive. On the other hand, there are cells that evade quiescence, and continue to proliferate. I believe that when this happens within a tumor (or spheroid), those cells are the ones that suffer from hypoxia. Moreover, unless they (i.e. hypoxic cells) escape the tumor (or spheroid), they will eventually die—hence the emergence of a necrotic layer.
As for your second question, what are your culture conditions? You mentioned chemicals inhibiting growth, are you adding any chemicals that may influence proliferation? If not, and assuming this is an in vitro study, then there could be a few factors that could be influencing spheroid growth. The first is the type of culture dish are you growing your spheroids in. The surface area and loading capacity of the dish affects spheroid growth. So therefore, just because the size of the spheroid is not increasing, does not mean that there aren’t proliferating cells present. I know this to be true because I have passaged spheroids to produce secondary and even tertiary spheroids.
Have you done any testing to determine the state of the cells within the spheroids? That is, what stage in the cell cycle they are in. You could dissociate a few of them and use flow cytometry to check the cell cycle. It’ll give you an idea of whether or not proliferation has ceased. Or you could passage them, and if secondary spheroids form, then at least you know that viable cells present and that proliferation is happening.
I hope this helps.
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I have 8 means for 8 concentrations inducing control
I calculated the proliferation %
How can I statistically analyze if the treatment significantly has an effect or not ?
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Is there a difference only in one variable/parameter (concentration) in cell culture experiments, i would propose to use One-way ANOVA analyses.
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I am doing my research on electrospun scaffolds for tissue regeneration and my team will have it them tested for biocompatibility and cell proliferation soon. I'm not yet sure which cell line will be used but I came across several papers that use cancer cells for such tests. I don't have a background on cell testing but I do know that cancer cells can proliferate indefinitely in a culture without the need for additional growth factors unlike normal cells which makes things easier. But how can it provide evidence of compatibility when my application is solely for normal cells (i.e. scaffolds for skin cell regen.)? I mean will the results be the same for normal version of the cell?
Please enlighten me about this. Thank you
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Dear Jean Raynell Bello
Cancer cells are immortal. Therefore, their use is unsuitable for assessing biocompatibility. According to the international standard, fibroblast cells are used for this task such as L929, SNL and etc. All biological tests must conduct according to ISO standards 10,993–5:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity). You can read the following articles:
Flexible magnetic polyurethane/Fe2O3 nanoparticles as organic-inorganic nanocomposites for biomedical applications: properties and cell behavior
Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs
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Are we suppposed to include dead cells that have not proliferated when calculating in vitro proliferation of T cells. If we keep only live cells we could be in a situation where the majority of the cells have died and the 10 live cells are proliferating thus we would have 100% if we consider only the live cells.
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There are probably exceptions but I would think that your in vitro assay would be invalid if you are getting the extreme case you are mentioning. You can remove dead cells from the % of CFSE-negative but ALSO report your % of dead cells. That would give evaluators of your data the most information. However, if you have high % dead cells - consider also adjusting parameters of your assay.
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Hello
I am planning to insert a mouse gene into dog genome via CRISPR cas9 to re-activate cell proliferation pathway. Not sure where to begin. If anyone who has done a successful CRISPR Cas9 experiment before, can you please share your experience with me e.g. how to set up the experiment? any help would be highly appreciated.
Many Thanks
Regards
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Kavita, Yes I believe HDR template is necessary along with sgRNA.
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Would you know of any study that measures any quantity related to the cell proliferation rate with regards to different concentrations in O2 or other nutrients ? For example the time of a cell cycle, or the growth rate K in dC/dt=K*C, or the probability for a cell to divide on a time T.
More precisely, are there single-cell experiments on that topic ?
Thx in advance
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Yes
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Is anyone working with immortalized human cardiac fibroblasts (abmgood) or primary human cardiac fibroblasts (PromoCell) ?
Do you have any problems with cell proliferating or spontaneus differentiation?
(I need to differentiate them info myofibroblasts and haven´t found any publications on this process.)
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Dear Veronika,
have you started to work with these cells?
As I would like to buy both of them, I'm looking for suggestions!
Thanks
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I am about to isolate CD138+ plasma cells using a Miltenyi Biotec MACS kit from the PBMCs of healthy donors.
Although I know that circulating plasma cells are scarce in normal individuals, I need to isolate them to use as control for primary malignant plasma cells that I am working on.
So, I need to know if I can successfully isolate plasma cells from peripheral blood of healthy individuals and the possible yields.
Thank you in advance!
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In normal person detection of plasma cell as an reactive cell especially in infective or inflammatory process is possible
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proliferated cells are harvested. How do I freeze them?
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Hi,
I prefer 1x10^6 as aseeding density for 1 vial and 1:4 or 1:6 passaging for confluent T75. As a freezing medium 90 % FBS and 10 % DMSO. I abstain from using growth medium addition to freezing medium.
Best wishes
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In several papers about the cell proliferation, generally they acquire not only BrdU(or EdU) results but also Ki67 results, and then compare these results. I want to know the reason why they acquire both and compare them.
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The Ki-67 protein is a nuclear antigen associated with cell proliferation and can be used as a marker for cell proliferation assay because it is expressed throughout the active cell cycle (G1, S, G2, and M phases) except for the resting phase (G0). The main advantages of assaying Ki-67 protein are that various techniques can be used: formalin-fixed paraffin specimens and frozen tissue samples by microscopy, single cell suspensions by flow cytometry, and cell lysates by western blot.
Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) can be used to label nascent DNA in living cells. During the process of DNA replication, BrdU can replace thymidine and incorporate into the newly synthesized DNA of actively dividing cells. Since the BrdU reagent does not cross react with endogenous DNA, cell proliferation thus can be reflected in the extent of BrdU incorporation by determining the absorption intensity of the final reaction. The BrdU cell proliferation assay is a versatile and convenient method for quantification of cell proliferation.
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Hi, I am trying to do in-vitro T cell differentiation from mouse naive CD4+CD62L+ T cells but there is something wrong that I cant figure out. I tried several times with different plates and antibodies but cells didn't proliferate at all. I coated the U-bottom nunc 96 well plate with 2ug/ml biolegend antiCD3e antibody (I tried with overnight 4 degree, 37degree or 2 hours 37 degree) and then I resuspended the cells in differentiating media that I prepared for Th1,Th2,Treg,Th17,Th22 with different antibodies. I am using complete media with 55mM b-merkaptoethanol, anti-CD28 concentration is 0.5 to 2 ug/ml. What could be the reason for cells are not proliferating or dying?
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I used to do differentiations during my doctoral work. You can find my protocol below. You would notice that I used to use soluble a-CD3/CD28. You could coat the plates with a-CD3/CD28 or just a-CD3 and then use soluble a-CD28. We have noticed that using 10% serum (instead of 5%) makes a lot of difference. If your differentiation works, you should see good numbers of large cell clusters in your wells.
Lymph nodes and spleens are obtained from mice. The tissues are ground and the cells are then treated with an RBC lysis buffer to remove the red blood cells. The cells are then passed through a cell strainer to obtain a single cell suspension. The naïve CD4+CD62L+ cells obtained from MACS are resuspended in RPMI supplemented with 10% FCS, penicillin / streptomycin, glutamine, HEPES and sodium pyruvate.
Differentiation is done on 24 well plates. 0.5-1 X 106 naïve T cells are plated out per well. The cells are then stimulated with 5µg/ ml anti CD3 and 1µg/ ml anti CD28 in the presence of cytokines. The cytokine concentrations used for are as follows: Th17: TGFβ- 2ng/ml, IL-6- 30 ng/ ml, IL-23- 15 ng/ ml, IL-1β- 10 ng/ml, aIFNg- 1µg/ ml; Th1: IL-2- 2 ng/ ml, IL-12- 10 ng/ml, aIL-4- 1µg/ ml. The cells are cultured for 5 days.
On day 5, the cells are replated in restimulation media (RPMI supplemented with 10%FCS, penicillin/streptomycin, glutamine) and restimulated with ionomycin (750ng/ml) and phorbol- myristateacetate (50ng/ml) for 4 hours in the presence or absence of Brefeldin-A (5 μg/ ml). The cells restimulated in the presence of Brefeldin-A are used for intracellular cytokine staining.
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I have a question concerning the classic MTS assay. I am performing MTS assays with HaCaT cells and can observe enhanced "viability" values after treatmenht (up to 30%). Assaying the cell proliferation by CyQuant assay, the DNA content after treatment is the same as in the control cells.
Does anybody have an idea, what my cells are doing with their "increased metabolism rate"?
Thank you :)
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Acceptance, Rejection, biocompatibility, cell adhesion, cell proliferation, etc.
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Hi all, I try to activate CD4-T cells isolated from PBMC using Dynabeads® Human T-Activator CD3/CD28 (Invitrogen) with cell/beads ratio 1:1 (1 million cells/well in 6-well plate). After 48 hours incubation, I collect cells and measure cell cycle by flow. It looks that my experiment don't work 'cause 99% cell still stay in G1 phase, like untreated CD4-T cells. Is there any trick in it? Any suggestion will be deeply appreciated! 
George
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I also failed to perform this experiment. I used positive selection T cell. I am afraid that that is the big problem for T cell activation. Can you share me more experiences?
Thank you so much.
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Unfortunately, the highest wavelength in my lab's spectrophotometer is 492nm with a reference wavelength of 620 nm. From what I read, most published paper used 570nm wavelength to measure their MTT assay. I was wondering if that would affect my reading?
My seeding cell density is 9000 cells per well and incubate for 72 hours, after 72 hours add 10uL MTT, and incubate for another 4 hours, after 4 hrs, discard all media with MTT, add 100uL DMSO for 1 hr and read the plate.
The absorbance came out in a range of 0.1~0.4 (cells+media), 0.05~0.07 (media only, background).
I am doing MTT to check the cell proliferation when growing in different media formulation.
Should I increase my cell seeding density to 10000 per well?
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It seems that in your case you get twice lower absorbance at 492 nm than you should get at 570 nm (where is the maximum absorbance of formazan, see the attachment). Reference wavelength is totally OK. It is not bad if you do not have other choice, until you measure the absorbance at the same wavelength in all cases including control.
Also it seems that you get really low absorbance (0.1-0.4) though you seed quite high number of cells (9000). Did you try to check through the microscope what was the confluency of your cells? If you seed too much, they may dye and then you get also low absorbance. I would suggest to do an experiment with different numbers of cells (2000, 4000, 6000, 8000, 10 000 cells/per well) and check which one will give you the highest absorbance after 72 h of incubation. Just always check the cell confluency through the microscope before the measurement. Good luck!
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I'm doing primary cell culture and I want accurate and more sensitive assay to deteremine the cell proliferation in real time. Since I'm using 3D cultute systems, I want an assay which should not get affected with the use of different culturing scaffolds.
-- Thanks in advance.
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The advantages of this method are: low to no cytotoxicity (depends on the cells you're working with), wide range of visualization options (infra-red, fluorescence, confocal, in vivo imaging etc.), high stability of the signal and relatively long time of action (we've succeeded to monitor the cells up to 24-30 days (again, depends on the type and the size of your cells). The signal can be quantified by the intensity and the number of foci.
If not, you could simply sample equal parts of the 3D culture, resuspend the cells in 2D culture and run an old good XTT/MTT assay.
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after 24-25 passages, I have realised that the cell growth rate decreased. there was not any contamination.
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Thank you very much Alex and Han! Is there any reference that you can suggest me to look because I can only find the HEK 293T cells are immortalised and no deep information about passage number related with proliferation.
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A gene become mutated and alter the cell proliferation and made a cause of cancer. So, how we can distinguish the is a oncogene or tumor suppressor gene.
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Dear Mohammad Ahmad,
the distinction is unclear, and it does not really make sense. Many genes have been moved from one to the other category over the years.
W.
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Conducting proliferation assay with H1395 cell line and treating them with different compounds in a 96well plate. The media used is 10%CSS phenol-red free DMEM. EGF is supposed to be a positive control but the cells with EGF treatment have the same amount of proliferation as the other treatments. Visual confirmation can be seen by observing morphology change of cells treated with EGF.
Steps: 1. Plate cells at 500,000cells/well, incubate in 37C till ~40% confluent
2. Change media with treatment media, incubate in 37C for 2-3days
3. Remove plate when near confluent, dump media freeze at -80C
4. Thaw at RT, then add 100uL diH2O, incubate in 37C for 1hr then freeze again at -80C then thaw at RT.
5. Used Molecularprobes fluoreporter dsDNA quantitation kit
(100uL of hoescht buffer in each well, then run in machine)
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Hi Tamor, your proliferation assay looks very strange to me.
500,000 cells per single 96 well? That is a lot of cells for a small space! Where did you get this assay from?
Beside the fact that most of the cells will probably be sticking to the sides of the well, you are selecting for cells that adhere quickly and may not be representative of the whole population. Most of them may be dead already if you didn't do trypan blue viability staining.
If this is a proliferation assay you are not allowing much proliferation by seeding cells at such high density.
The rest of your assay doesn't make sense to me.