Cell Proliferation - Science topic

I have observed IFN-gamma induced growth inhibition in few of my cancer cancer cell lines. I assume that is due to the activation of STAT1 phosphorylation which in turn is targeting some downstream effectors of cell cycle arrest. To understand the exact mechanism, I want to inhibit STAT1 phosphorylation without interrupting the cell proliferation. Can you suggest any pharmacological inhibitor which will not kill or toxic to the cells itself?

Its an In Vitro Cell Proliferation using Leukemia patient cells lines. Cell Proliferation Dye Used : Tag-It Violet Cell Proliferation Dye. Thank you a lot.

Telomere length has been recognized as one of the best biomarkers of aging, indicating its importance in understanding the aging process. However, the evidence suggesting telomere length as a definitive biomarker of aging in humans is not conclusive and remains equivocal. Staying in harsh environments like space stations can lead to telomere lengthening as an adaptive response to stress and radiation exposure. Research on astronauts and individuals in high background radiation areas has revealed interesting findings regarding telomere dynamics in such conditions. Stressors such as space radiation and microgravity can trigger telomere lengthening, which typically reverses upon returning to Earth. This phenomenon is influenced by factors like radiation dose, dose-rate, and radiation type, underscoring the complexity of telomere modifications in extreme environments. Similarly, residing in another challenging setting, such as an underwater compound in a Florida lagoon, may also result in telomere lengthening due to increased pressure and exposure to environmental stressors. The compound's atmospheric pressure is 70% higher than at the surface, impacting bodily functions such as urination and metabolism. Researchers like Dr. Joseph Dituri are investigating the prolonged effects of this heightened pressure on the human body, as it could potentially offer insights into reversing the aging process and extending lifespan. The elevated pressure is thought to boost stem cell proliferation, telomere length, and collagen production, potentially slowing down or reversing aging effects. Nonetheless, it's crucial to recognize that continuous telomere replenishment is a hallmark of immortal cells like cancer cells, necessitating further research to grasp the possible consequences of these transformations.

I welcome your comments. References: [1] https://lnkd.in/gRkczjVf [2] https://lnkd.in/gPQxpbCW [3] https://lnkd.in/gWDpfnXk [4] https://lnkd.in/ghipmQnp [5] https://lnkd.in/g7JcAQmN [6] https://lnkd.in/gbMZmn_Q

Hello everyone

I am trying to design an MTT experiment for an immortal adherent cell line with doubling time of 20 hours. The goal is to evaluate effects of certain growth factors and their combination on cell proliferation and determine the optimal dose.

Many papers suggested seeding 10000 cells in 96 well plates and performing MTT at 24 h intervals or at days 1, 3, 7.

I am not sure about some technical issues:

1. should I proceed with 24 h interval assay and for how many days? or the 1, 3, 7 days evaluation is good enough?

2. can I seed 5000 cells/ well in 96 well plates instead of 10000?

3. I expect my cells to become confluent after 72 hours (starting from 10000 cells), and they definitely die without medium change, so should I change the medium every 24 hours for all plates? or just change the medium after 72 hours and wouldn't this affect my results?

Thanks a lot!

I am working through the challenges of cell isolation from the rat heart. I am using VSMC media and endothelial cell media. In a homogeneous mixture of cells, would these media favor only the growth of the 1 cell type that they are optimized for? For example, I know endothelial cells require specific hormone factors which other media lacks. Or would other cells proliferate just as well in these media? For example, vsmcs in endothelial cell medium or fibroblasts etc.

Hi

I want to check the cell proliferation using live/dead cell imaging technique till 72 hr. What should be the cell seeding at the start if using 24 well plate.

Thanks for your time.

I am now studying a protein, this protein might affects cell proliferation because I performed a Knock down experiment and I found that the cells proliferation rate is reduced when this protein is knocked-down. This protein is a trafficking protein which has role in transport from ER - Golgi, I suspect that the mechanism related to this reduced proliferation rate is because of the delayed cargo shipment to golgi. What method is used to identify the cargo protein of this protein ? I want to collect many materials before I spoke to my PI

Hi,

I'm using CRISPR to knockout a gene. The two gRNAs that I used were different in their efficiency in reducing the protein expression level (one reduced over 95% of the protein, the other reduced only 60%); however, these two gRNAs were suppressing cell proliferation at the same level. Could anyone think of any explanation for this?

Thank you!

Lei

Planning to run my assay this week but couldn't figure out positive control for MTT assay. I want to use cells without treatment as negative control while needing an agent or growth factor that induces endothelial cell proliferation as positive control. Please your advise on this will be appreciated. Thanks

While culturing immortalized ameloblast lineage cells from frozen stock (liquid nitrogen), despite there being good attachment numbers, subsequent proliferation of the cells is slow to negligible. These cells have a tendency to proliferate quickly when seeded with a moderate cell density. Previously, we were able to successfully culture these cells under identical conditions (low glucose DMEM enriched with 10% FBS and antibiotics). Currently however, the cells do not attach to plates unless high glucose media is used. And the attached cells are not proliferating despite a good proportion of cells with tight cell-cell contacts. Any suggestions on what other parameters to change are much appreciated! Thanks!

1) What all mitogens can be used to increase cell proliferation apart from classical/mostly used growth factors to enhance cell proliferation?

2) Can this be universal for all cell types?

I culture multilayered cells, and I found previously that the live nuclear staining dye Hoechst 33342 enters the cells not in contact with the culture medium (cells in the lower layer) very poorly. I am afraid that BrdU would also fail to enter the cells, and I would get a false readout for non-proliferating cells. What other markers (immunofluorescence based) can I use to detect cells that are likely to divide (S-phase)?

My protocol requires the establishment of clonal cell lines through the expansion of single cell clones sorted in individual wells by FACS. This is notoriously challenging for fibroblast cell lines such as BJ, which often do not proliferate as isolated cells. Do you have tips for stimulating fibroblast cell proliferation after single-cell sorting?

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.

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

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)

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.

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!!

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.

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.

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?

Because I want to make B16F10 cells proliferate faster in vitro, I wonder if 20%FBS will be better. Thank you.

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?

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

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,

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?

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?

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.

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)

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

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?

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?

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.

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.

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.

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.

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.

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.

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)?

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?

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?

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

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?

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

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.

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.

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!

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.

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. :)

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?

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.

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!

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 :)

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

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!

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?

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,

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.

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?

This question is similar to a previous one https://www.researchgate.net/post/What_is_the_difference_between_mechanism_of_immortality_in_normal_stem_cells_vs_cancer_stem_cells

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

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.

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?

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.

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....

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?

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)

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 ?

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

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.

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

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

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.)