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Cell Line Culture - Science method

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I am currently culturing NK92 cell line in complete RPMI supplemented with 500u/ml IL-2. Observing steady-state cells by fluorescence microscopy, I observed that the majority of them have a rounded but sometimes irregular shape, with a single nucleous showing lobes. However, about 10% of them showed a more regular rounded shape and a perfectly spherical nucleus.
Since this morphology correlated with the expression of a protein of interest, I was wandering if someone could tell me if the "more regular" shape could correlate to any functional state for these cells. 
thanks!
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Hi , I think it's a good idea , recently I culture NK92 cell line , and find the same situation , I advance you can extract some protein such as NK activiated or inhibitory receptors , compare them maybe find some interested.
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Hello everyone,
According to the analysis of cell-surface markers using flow cytometry, is it possible that trypsinization would block or even digest the markers?
If so, based on your experience, what is the best way to dissociate the adherent cells from the flask? Thanks
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The quality of analysis in terms of detection of surface markers using flow cytometry will be determined by the cell detachment method.
The commonly used methods of adherent cell detachment include enzymatic and mechanical treatments. Enzymatic detachment, however, can lead to significant changes in cell membrane protein structure and composition leading potentially to significant experimental bias.
Accutase which is an enzyme mixture with proteolytic and collagenolytic activities is usually considered a less damaging agent than trypsin and is recommended for the treatment of sensitive cells as well as for analysis of surface markers using flow cytometry.
On the other hand, detachment by the mechanical method is achieved using the so-called ‘rubber policeman’ which is a rubber or plastic scraper attached to a glass rod. But again, mechanical methods can lead to cell membrane damage and to substantial changes in its structure.
Thus, choosing an inappropriate detachment method may cause unintended effects biasing experiment results. So, I would suggest that you conduct a pilot experiment to assess the optimal cell harvesting method (either accutase treatment or mechanical detachment using a cell scrapper) for your cells to avoid experimental bias and obtain reliable results.
Best.
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The Boyden chamber protocol requires a high budget for us. We want to try modifying this test instead. Can we combine it with a protocol like the filter diffusion protocol?
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If you are looking for a substitution for this experiment maybe you can try Zigmond chamber. It's resumable and can be used to monitor cell migration in a real-time manner
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Hi Everyone,
I am culturing the primary cells (Monocytes) from the patient blood samples. During the culture (2-3 weeks), I have seen the long tape shape black color filament (1 or 2 in number). Is it a type of contamination? How to overcome this?
Thank you in advanced.
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Doesn't seem like a contamination, but more of a poly(propylene)fiber filament, a result from plastic injection of pipette tip during production. We usually ignore it, through centrifugation it will disappear, eventually. Else keep an eye on the tip, if one showed with filament seems going to detach, please discard it and choose a new one.
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Hi Everyone,
Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?
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I aslo think that checking the purchaser information for cells/media would help. And, addition of L-glutamine to medium already containing l-glutamine is mostly not necessary.
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Hello,
I am working with M93-047 cells and I have cell lines cultured in T75 Flasks, I want to seed cell for Pull down IP experiments. I want to seed cells either in 3 or 4 10cm plates, so that I can have 3 or 4 samples of the cell line to work on.
I am confused that what dilution of RPMI media I should use to seed M93 cells, so if someone can help, and then secondly after counting the viable cells using hemocytometer, what factors we should keep in mind for seeding.
If someone can share good protocol for seeding cells, I'll appreciate.
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Thank you so much for answering my question, it’s a big help.
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Hi everyone,
I have seen some very strange debris in my U87MG (glioblastoma cell line) culture. It seems that there are shards/fragments of glass that are not contaminating the cell culture and the cells are potentially trying to adhere to them. I attached some pictures of the debris, taken shortly after passaging, so they are not fully adhered.
When I first saw this, I bleached the cells, threw out the package of flasks I was using, made new media, etc. I figured that this originated from a glass pasteur pipette, so I thawed another vial of cells that was frozen down at a different time than the previous ones I used. However, I'm still seeing the same debris in the freshly woken up cells.
I'm confused because I didn't see the debris in the first set of cells until weeks into using them, and I didn't see them in the second set of cells until about two weeks since I thawed them. I may have just not seen it until two weeks in, but it just seems unlikely due to how much and how obvious it is.
This is the only cell line I use EMEM (w/ penstrep & 10% FBS) for, and I have never seen this in any of my other cell types that use other media (completed with the same batch of penstrep and FBS).
Please let me know if you have ever seen anything like this before! Any comments are appreciated - thank you all in advance.
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Hello Nicole,
I sometimes saw them after defrosting the cells. Such structures are formed by DMSO if it is unevenly mixed when cells are frozen.
When adding to the freezing medium, try to stir the DMSO as vigorously as possible. You can also add DMSO directly to the cryovials containing media and cells and mix thoroughly. A small volume of DMSO is easier to mix.
Good luck!
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What is the time taken to reach confluence for hepg2, huh7, heap 16 cell lines cultured in 10%FBS, DMEM high glucose
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I am trying to induce inflammation in the cell lines (Beas 2B & NHBE) by using the dust but haven't worked with cell lines previously. Kindly help with how to identify the cell lines whether it is inflammated or not?
What are all the parameters I have to consider?
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Hi Deepdarshan.
BEAS-2B is a human lung epithelial cell line that shows prominent inflammatory responses after induction with Dust. As I am working on Fine particulate matter induced lung inflammation So I would suggest you to plate 8-10k cells per/well in a 96 well plate and check for the cell viability with varying doses of dust particles treatment. Further you can choose the most significant dose where cell viability is reduced and there is an increased expression of inflammatory proteins and an decreased expression of anti-oxidant proteins (can be done by immunoblotting of immunofluorescence). You can also measure the ROS by CM-H2DCFDA or MitoSOX to conclude the oxidative effect of dust particles in lung epithelial cells.
Thanks
Hope it helps...!!
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Is it ok to grow GRANTA-519, with RPMI-1640+10 %FBS?. When I tried then found that these cell lines are also growing within this media.
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How does the passage number of a cell line affect experiment results including toxicity assays? Which characteristics of cells are changing as the passage number increases?
What is the most efficient or optimum passage number of cells (for example, for cancer cell lines such as HepG2, A549 etc. or for healthy cell lines like HEK293) for setting an experiment?
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Passage number as the number of times cells have been transferred from vessel-to-vessel, which affects a cell line's characteristics over time as the passage number increases their phenotype and genotype can change. Hepg2- Better to do within 16 passage, A549- Better to do within 20 or 30 passage, normal Hek293- Better to do within 20 passage.
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I've been trying to differentiating SH-SY5Y cells with brain-derived neurotrophic factor (BDNF) in 8-well chamber slides but I seem to be having problems with cells detaching over the course of the 7 days in serum-free media with BDNF. The problem seems to be more apparent after changing the media but I am unsure as to why this would occur and would value any insight from people with similar problems with SH-SY5Y or other cell types. My first thought was that I was removing too much media from the well, so I began to leave around 1/4 of the old media and add 3/4 new media and pipette the media off very slowly and add the new media very slowly (to the side of the well, not directly on to the top of the cells) but I still seem to be having the same issue. I thought there might be a problem with the incubator I was using so I changed incubators, and yet the problem persists.
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Did you coat with PLL?
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Wondering if TrypLE Express cell dissociation agent is significantly better than other options for culturing mammalian cell lines. Currently mainly using Accutase (for fibroblasts and epithelial cells, NOT stem cells) and sometimes also trypsin for specific cell lines. Any positive/negative experience with TrypLE Express enzyme? Main attraction is that it does not require neutralization as trypsin does, but has longer incubation times. Would appreciate any comments and suggestions.
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Yes, you can use TrypLE Express for cell detachment. I used all mentioned dissociating reagents for cell detachment.
There are other benefits such as:
(1). Its exceptional purity increases specificity and reduces damage to cells that can be caused by other enzymes present in some trypsin extracts.
(2). TrypLE Express remains stable for 24 months at room temperature or 2–8°C, making storage and handling easy and convenient.
I treated cells with this enzyme, in the same way, I did with trypsin EDTA or Accutase, and found no significant difference in detaching cells from the cell surface.
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I thawed a tube of Calu-3 cells from ATCC, following product instructions. The flask I used was not coated with any substrate. I used EMEM with 10% FBS and placed it in the usual 37degC incubator with 5% CO2 and water pan. The next day I discarded floating cells. Since then, these cells have always appeared round and bright (see attached photomicrograph). They appear to be able to change positions, moving from middle of vessel to the sides. At first the numbers grew slowly but later it started dying. I have changed the media to DMEM with sodium pyruvate and 10% FBS. I had transferred floating cells to another vessel coated with collagen IV. I had also trypsinized one flask and transferred all cells to one collagen IV-coated well of a 12-well plate, and refreshed media every 3 days. Each day I waited for it to adhere firmly and resemble the clumps of cells I saw in the ATCC photomicrographs. But after a month, they are still round and bright. Now, they are starting to die off. Can anyone advice or suggest what I can do to rescue these cells?
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We coated our flask/plate with 10ug/mL collagen IV. Is this concentration too low compared to your protocol for substrate coating to support growth/adherence?
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I recently sequenced (Sanger) an exon from single alleles of a CRISPR-Cas9 mutated cell line (diploid) to find whether the mutation is homozygous or heterozygous and what the mutation is. What I got is 3 different results, a WT, and two diff mutations, a 1 base del and a 7 base del. My lab has come to the conclusion that it must somehow have 3 alleles instead of 2, and suggested looking into this. I can't really find anything relevant to this find, so I'm wondering if anyone might know of such research articles or point me in the right direction, and if this is even possible?
My original thought was that perhaps the 7 base deletion is somehow an error due to the crispr process? But more experienced people than me have suggested it means there are 3 alleles, although like I said I'm struggling to find relevant literature to support this.
Any help would be much appreciated, thanks.
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My thought is that you have a mixed population of cells, each with their own alleles. Streak/dilute them out to single cell colonies and screen them again.
No, your cells are not magically triploid.
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I have difficulty every time whenever I subculture C6/36 Cell line and vero cell line.
Can anyone suggest how to spread cells evenly in growth media. 
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How many Vero cells are in one T75 flask
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Kavitha Jade Brunner it sounds to me like you're doing the right things regarding the water in the water baths. As for cleaning surfaces, using the bleach wouldn't cause contamination, it can just wear down on the surfaces over time.
Disinfecting the scope and surrounding areas as best you can will likely help quite a bit! I'd love to hear if it helps as I am quite curious as to your contamination source as well, now.
I'm not sure why you don't filter media, but perhaps there is something about these cells/this media that I'm unfamiliar with, which is certainly a possibility! Since you're using antibiotics, it can be good to regularly test for mycoplasma as well if you don't already. Though you may not have visible contamination in all your flasks, contamination can sometimes be masked by antibiotics. It seems to be a point of contention among scientists (much like "a-POP-tosis" versus "a-PUH-tosis" haha) but I personally prefer to culture cells without antibiotics. If your aseptic technique is good (sounds like yours is - your source of contamination is likely from the dissection conditions), there's really no need for antibiotics in my opinion! Your PI may vehemently disagree, but that's just my two-cents. Good luck and feel free to keep me updated as to whether the scope disinfection helps!
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In my experiments, I found that different monoclonal cell lines from the same host cell produce similar concentrations of ammonium ion when using the same medium; but when the same monoclonal cell line cultured with different types of fermentation medium, the concentrations of ammonium ion vary greatly. Is it because which of the ingredients in the medium is different that makes this difference?
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Hello Xukun Xu
The components in the medium affect the concentration of ammonium ions. Thus, to minimize both lactic acid and ammonium production, glucose as well as glutamine may need to be simultaneously controlled or replaced. You may refer to the article attached for more information on this subject.
Best.
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Hello everyone.
I am looking for suggestions for HL60 cell line culture. Our lab is new to suspension cell culture. we bought the cell line from National Center for Cell Sciences (NCCS) Pune. Upon arrival, the cell was absolutely fine, having a doubling time of around 48 hours and we were able to freeze 10 vials with 10^6 cells/ml (90% FBS + 10% DMSO). But after 6 months in liquid Nitrogen when we started culturing from those vials it seems that the cells are not proliferating. We are seeing cell clumps forming and after 3-4 days all of these cells are dying (when checked using trypan blue). Can you help me with this problem? We are unable to point out the exact cause of the situation.
Thanks
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Thank you all for your reply. Yes, María Luisa Caballero we have frozen the cells slowly at -80ºC then passed into N2 after 3-4 days. One thing I would like to add is that we are using "adherent or treated T25" flasks. Is that be the main cause?
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I just got a new stock of NR-6 cell line (Mouse embryo fibroblast cells that don't express EGFR) and I need more information about the culturing and specifications of it.
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The above described the culture media. The best is to ask the company or lab you got the cells. They are the ones who culture and make the stock. Alternatively, you can search using PubMed or another. Good luck.
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I want to know the expression change of my target protein by western blot from the control and drug-treated cells. 48hrs. after the treatment, I have pelleted the cells and extracted the proteins using RIPA buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 2% NP-40, 1% SDC, 0.1% SDS, 2mM EDTA). After denatured the proteins using SDS-PAGE loading buffer, I have loaded the samples in SDS-PAGE wells. But, some of the samples leaked from the resolving gel and it did not stack properly. The samples spread laterally as soon as they entered resolving gel. I have prepared fresh RIPA also, but the result did not improve. As a control, I have run my old extract in the same gel. but it did not leak. My labmates use the same reagent, but they do not face any problems. Previously I have thought that it may be due to sample overloading so I have reduced the volume. But it did not seem the actual cause. It would be very helpful if anyone inform me of the root cause of this type of sample spreading in the gel. Please find the images, attached below.
Thank you
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SDS-PAGE gels have a limited shelf life, in an air-tight container wrapped in wet tissues about 2 weeks. Disassemble a casette to check that the gel is firm and well formed.
Also, I'd reduce the salt concentration in the homogenisation buffer to, say, 50 mM.
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Hi everyone, I am doing 2D monolayer cell culture on HeLa cells and A549 cells purchased from ATCC. I have had some problems after a few passages that cells occasionally all become apoptotic-ish looking. The cell confluence is normally high in that case, and instead of a more elongated morphology like what they normally look like, the cells can become round and flat (I cannot distinguish the contrast between nucleus and cytoplasm using bright field anymore) within one or two days. I have attached a picture of the HeLa cells that went wrong/abnormal looking below.
The media I am currently using is DMEM Phenol red free.
Can someone please provide some suggestion on this wether it could be caused by infection, medium related issue, or related to sub-culture procedure.
Thank you very much!
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Some suggestions:
1. May be passage them when they are 60-70% confluent?
2. Check the passage number? I know they say you can keep these cells growing forever but you could try a different batch?
3. Remove the DMSO completely when you plate them from stock. I used to thaw cells by adding 1 ml warm media drop by drop while holding quite tightly in my hand and then mix them to get a single cell suspension. Next, add them to warm media, centrifuge at 200 g for 5 mins, then plate the pellet in fresh media and off yoy go. Residual DMSO can cause some damage to freshly thawed cells (unless you change the media within 18-24 hours)
4. I use RPMI media, 10% FBS, l-glut, gentamycin.
5. Check whether the media is high or low glucose and see which works best.
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I am using G418 to select for stably transduced 22Rv1-luciferase expressing cells. Would anyone know of the minimal effective concentration of G418 required to kill 22Rv1 parental cells? I am currently using 1 mg/ml in my media, but I'm not seeing much cell death.
Thank you!
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Yes it is very inefficient for some cell types. Requires very high concentration for some cells.
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I'm working with BJAB lymphocytic cell line, culturing them with RPMI media (with L-glutamine) + 10%FBS + 1% antibiotics. After two days of changing media by centrifugation, they started to adhere to the T flask and a monolayer has formed. Does someone know what could be happening? Is the media lacking something or should it be a step in the handling that I'm missing?
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Sandra Cortes you should check the type of culture flask first that should not be tissue-culture treated one, and maintain cell culture with requires agitation (i.e., shaking or stirring) for adequate gas exchange. Try this maybe this can help you.
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I'm focusing on L929 cells to collect the supernatant using for Bone-marrow-derived macrophage (BMDM) cell culture.
Our L929-Conditioned Medium was out of stock and I started thawing an old L929 stock (in -80°C fridge) (it is written 2014 but maybe even before that time).
I followed the protocol of our lab to grow the cells and collect the supernatant.
Our lab used that protocol for very long time and it still worked well.
However, now the BMDM cells which I cultured by using that supernatant grew not as well as normal, the morphology changed.
Then, one of my senior who has been working long with L929 cells said that my L929 cells after 100% confluent continue growing too strongly, which forms several layers of cells.
She said it was abnormal because based on her experience, after 100% confluent, L929 cells will be inhibit somehow to hardly multiple as many as that.
(The L929 supernatant stock I used before was from her and It did work well, now I started thawing new one. )
That might be the reason why my L929-conditioned medium was so poor in nutrition, and might be also the reason why my BMDM growth medium didn't work well as normal.
We think it is because of the degeneration of L929 cells.
Does anyone have any experiences with this? Please let me know. Thank you so much.
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dear Anh
regarding the L929 cells I prepared it so many times during my PhD and it was working very well. I usually use Triple layer flasks to have a good amount of the supernatant. In your case you can do a flow cyometry for macrophage markers F4/80 and CD11b in order to convince that all your cells are macropages not other cells. I would also recommend using less passage for L929 cells no more than P5.
Kind regards
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I am trying to culture dental pulp stem cell line from our storage. The cell line was preserved on 2019. However, the cells aren't adhere to the flask. I used DMEM with 20% FBS, 1% penicillin and streptomycin, 10mM L.Ascorbic, 1mM L.Glutamin. I tried different cryovial from our storage but none of them tend to adhere to the flask.
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It seems that there is some issue either with the cell line or the culture flask itself. Did you check the viability of non-adharent cells in the flask after 24 hr of seeding? Did you find any cell adhared to the flask? If no, kindly get one vial from other sources to rule out any issue related to the cell line storage/damage. You can also check the final pH of the culture medium. With this, hope you may get the issue resolved. Good luck
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Gentlemen,
We are preparing a project to access a grant, but we are missing a lab that can help in the first phase of Proof of Concept and Safety.
The lab should be European (But not spanish, and sorry, but not UK) and be seasoned in brain samples handling, and also if possible having experience in cell morphology study (Neuron, Microglia, Astrocytes) or metabolic study (on said samples)
The project includes the development of a protocol in humans but we need to try it before on brain samples, and we are in a hurry to find a lab that can provide experience in this field.
Since we are in a hurry, you might contact me directly or respond in the thread.
Sincerely, J Vigil
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Please have look on our(Eminent Biosciences (EMBS)) collaborations.. and let me know if interested to associate with us
Our recent publications In collaborations with industries and academia in India and world wide.
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I want to do some cell migration assays and I would like to know if it's possible to grow these cells in suspension
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Hi
Saifun Nahar
, we are cultivating ID8 cells with 10% FBS. It will not cause them any problem BUT! Different % of FBS can cause differences e.g in gene expression and other aspects. I would make sure to do ALL experiments with the same % of FBS so you will avoid differences between results. I hope it helped, have a nice day :)
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Suppose i got a cell line say for example (CHO) and i want to maintain it for my experiments, I'ld like to know what all the properties/characteristics I've to know about the cell line, I'm thinking few points like :
Doubling time, It's morphology, To know whether it is adherent/suspension cell line, It's Suitable media.
I'ld like to know what all other things I should know ?
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You will receive a product sheet with all of the cell lines you have purchased, which contains detailed information on how to handle the cells. The product sheet also includes batch-specific information like the number of cells per vial, the recommended split or subcultivation ratio, and, if available, the passage number. Prepare the appropriate medium, serum, and other reagents for reviving cell lines by assembling the necessary growth medium, serum, and other reagents. Many of these items are available from suppliers and can be combined with cell lines to create a custom order.
After receiving your desired cell line, whether adherent or suspension, thaw the cell as directed on the product sheet, count the cells using a hemocytometer, and reseeded into a culture flask based on the cell count. Finally, examine the culture flask under an inverted microscope the day after it has been thawed to look for any anomalies.
A new cell line is being studied. Even if it has been done before, it is better to repeat the procedure by performing a simple karyotyping test because there is a chance that some important characteristic of the cell has been lost due to prolonged passaging.
If the cell characterization has been passed, then propagate your desired cell line and prepare for the stock by using the recommended freezing media according to the product sheet of the supplier and transferring the frozen cell line into the liquid nitrogen vapor phase as early as possible.
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I want to add rat tail collagen (type 1) to the DMEM medium for an in vitro animal cell culture experiment. Suggest me an alternative for dissolving collagen other than acetic acid.
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I am passaging HEK 293 cells for retroviral transfection.
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Passaging is required when cells have proliferated to confluences around 80%
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I am doing a cell culturing experiment in a fluid flow chamber made of PDMS material. I was wondering how to sterilize the pipes and the flow chamber before/after the experiment, in case I want to use the same chamber for the second and third biological repeats?
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Hi,
We used devices repeatedly made up of PDMS as well as other materials with pathogenic bacteria. As long as the materials are Autoclaveable there is no problem in reusing them. Infact the current scenario of having sustainable development contains inherent clause of showing how we can use the same devices for longer periods with use of minimal processing. Apart form that, technically PDMS has shown to provide good results as long as the devices made from it are sterilized meticulously. It all percolates down to how good your skills are while handling cell cultures!!!
Regards
Amit
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Using the recommended media and temperature to grow the cells but they die following the first sub-culture. When revived from frozen they look very healthy but as soon as they are split most of the cells die. Have tried different batches of FBS to no avail. Does anyone have any ideas?
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Lindsay M Maclellan I solved my problem with hFOB 1.19 culturing them in ploly-L-Lys pre-treated plates. And the cells grows so fast at 37°, even reach almost 100% of confluency in 5 days.
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Hi everyone,
I have been culturing MC38 cells for a few months and I am experiencing some problems. The cells aggregate a lot and generate clumps on the flask surface. I am using Trypsin to split them but it is really hard to de-attach them once they have created these clumps. I started splitting them 1/10 every three days and then switch to 1/20 to see if it improved. The viability tends to be low as well, around 50-70%. I am using DMEM+10%FBS as culture medium. If anyone has any suggestions and recommendations on how to culture these cells it would be really helpful. Thank you
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To break the clumps into single cell you can use vigorous pepetting.
Wash the cells twice with 1x PBS before trypsnization.
The moment your cell became single while seeding, you will get rid of this clumping problem.
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I am currently culturing some HEK cells with absolutely no issue, they are being cultured with the standard DMEM + 10% FBS media.
For a particular experiment, I need to introduce exogenous insulin for a variety of different time points and I am noticing that my cells are dying within 5-10 min of insulin addition.
I am adding insulin in at a concentration of 10ug/ml. The cells are grown in a 6 well and all those that dont have insulin added to them are surviving but the addition of insulin is resulting in all the cells lifting within 5 minutes. If someone could provide some guidance.
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Dear Smarth!
Plese You look a t the article
Perhaps insulin through its receptors on HEK293 cells enhances the accumulation of APP, which is toxic to these cells
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I tried staining MCF7 and MB231 for Vimentin and Cytokeratin antibodies, and surprisingly, I saw both cell lines showed both markers. According to the literature, MB231 is a mesenchymal type cell and MCF7 is luminal, so shouldn't it be that MB231 shows more cells positive for Vimentin as compared to MCF7?
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Hi Rafał Watrowski . Adity Pore was just asking why markers for the hypothetical EMT do not make sense in the 2 cell lines she is looking at.
I agree. The EMT is just a hand waving explanation of how epithelial cells, that are normally sedentary, suddenly become motile and invasive when they are cancerous.
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Hello,
Recently I thawed couple of cell lines, RPMI8226 and JJN3 and they both didn't do well during my first passage.
I thawed the vials in 37 deg water bath for couple of minutes. Transferred the cells with media into a conical tube. Centrifuged the tube at the lowest speed for 5mins. Aspyrated the supernatant and resuspended the cells in 10mL media in T-75 flask for 3 days.
I use the following media:
Gibco RPMI 1640 with L-glutamine + Penstrep + Glutamine + 10% FBS
My percent live cells was very low when I checked them 3 days later. I am thinking of using 20% FBS next time I thaw cells. Is there anything else I can do differently to keep the cells alive?
Thank you!
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Hello,
As Joseph mentioned, you have to be careful for how long you´re warming the cells, thawing freezed cells has to be a fast process, since DMSO is toxic for cells and can be the reason why you have low percent of live cells. Remember to check for any thawing specifications regarding your cell lines with your supplier.
Basically, both Freshney´s Culture of Animal Cells textbook and Thermo Fisher Scientific´s webpage recommend the following general thawing protocol:
1) Remove the cryovial from liquid nitrogen or -80°C freezer and immediately place it in a 37°C water bath. 2) Swirl the cells until you see a small bit of ice in the cryovial (this shall take about one minute). 3) Transfer the cryovial to a laminar flow-hood, open it, and transfer the thawed cells to a centrifuge tube. 4) Add pre-warmed medium appropriate for your cell line in a dropwise matter. 5) Centrifuge the cell suspension at approximately 200 g for 5-10 minutes (this vary with your cell line, I recommend you to check). 6) Decant the supernatant, resuspend the cells with medium, and transfer to a culture flask.
Note: some cells can be thawed without the centrifugation step, you can dilute the cryoprotectant with fresh pre-warmed medium, but you must change the medium the day after thawing.
Suggestions: make sure to work fast but aware of the steps of the process; while thawing the cells in the water bath make sure not to submerge the cryovial since this can increase the probability of contamination; try not to thaw cells that were freezed a long time ago, since viability can decrease; remember to always work aseptically; double check the label of the cryovial to make sure of the identity of the cells; plate thawed cells at high density to increase recovery.
In case you want to read more about thawing, Freshney´s textobook is very useful: Freshney, R. I. (2006). Basic principles of cell culture. Culture of cells for tissue engineering, 3-22.; you can also visit Thermo Fisher Scientific website, which has a video and a guideline regarding the thawing process: https://www.thermofisher.com/cr/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html
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I am culturing NK3.3 cell line. I do not have any experience with them. Unfortuately, they do not grow and I do not know why.
I am using AdvancedMEM media, 10%FBS, 1%P/S, 1% glutamine and I add IL-2 separately.(I aliquoted IL-2, so I heat my media in water bath, také out how much I need and add IL-2 directly there to avoid degradation of IL-2 while it is in the water bath. I dont know if this is the best solution?). I use100U/mL of IL-2.
When I count my cells, it has never been more than 250 000 NKs there (its been three weeks already, I should have more!!). When passaging, I leave 600mL of old media and add 2mL of new media with IL-2. I put the cells so that T-25 flask is "standing" with the cap up.
I passage them 2x per week. Whenever I count them there are quite a lot of dead cells visible.
Any idea on what am I doing wrong? Any suggestions/advice would be amazing.
Thank you so much!
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Dear Hana!
Mode of Growth:
  • Cells grow in suspension. Feed cells every 3-4 days. Cells are maintained in T25 or T75 flasks kept upright in the incubator with 6% CO2. The upright position favors cell-cell interaction, which appears to help them grow.
Method of Passage:
  • Draw off the half to 2/3's of old medium without disturbing the cells at the bottom of the flask. Resuspend cells in the rest and count. Resuspend cells in fresh, warm medium at a concentration of 3 x 105 cells per ml. Do not have a total volume exceeding 20 ml in a T25 or 50 ml in a T75 flask.
Culture Medium:
  • RPMI 1640 (pH 7.2)
  • 15% heat inactivated fetal bovine serum (FBS)
  • 15% PHA-stimulated PBL supernatant as IL-2 source (e.g. Lymphocult-T from Biotest, No. 811010)
  • 2 mM L-glutamine
  • 10-25 mM HEPES
  • 2 g/l Sodium Bicarbonate (NaHCO3)
  • 50-100 IU/ml Penicillin,  50-100 µg/ml Streptomycin
  • Try this protocol.
  • You said that you put the bottles vertically with the lid up, usually the bottles are placed horizontally in the incubator so that the surface area that is washed by the medium is larger, then there will be more cells. Even when transplanting cells, it is necessary to prepare a new medium with serum and IL-2 each time, and not to take the used medium and add 2 ml of new medium to the tube. In the used (conditioned medium), the cells metabolism cells accumulate, they inhibit cell growth.
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I searched a few papers but nowhere could find the answer. Asking out of curiosity. I went back to the first few papers that talked about RAW 264, RAW 309 cells etc. also I checked on the official webpage of Abelson's Lab from NCI, NIH. I couldn't find the answer. 
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Named after the three authors on the original paper
Ralph, rAschke, Watson
  • DOI:10.1016/0092-8674(78)90101-0
Corpus ID: 5529220
Functional macrophage cell lines transformed by abelson leukemia virus
  • W. Raschke, S. Baird, +1 author I. Nakoinz
  • Published 1978
  • Biology, Medicine
  • Cell
Abstract Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing…
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I had a contaminated cell culture I suspected several things, including the media. I put the media (only) in a T-25 flask and used 2 agar plates to check bacterial contamination (one opened in the laminar air flow the other in the CO2 incubator) and left them is in the CO2 incubator at 37 oC (after I cleaned it),
two days later I didn't find any thing on the agar plates and no changes in color or clarity of the media in flask however I examined the flask under the microscope and found these things .. What could it be?
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I understand that this can be very frustrating. The other answers are excellent responses for your problem. It is interesting that you had no contamination in your T-25 or agar plates.
I have found that without a cell layer to guide you, it is quite difficult to locate the media in a flask. Are you absolutely sure that you were looking at the media and not the bottom of the flask - the patterns in your picture resemble scratches and divots on the outside of the flask.
In any case, to help prevent contamination in your media, you can aliquot into 50 ml tubes and use those instead of an entire bottle at the same time.
Hope this helps!
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I have to treat cell line for 6 days with compound to see its deleterious effects. But the thing is within 48 hours cells got confluenced. So how to do this experiment? Should I just decrease serum concentration or any other way to complete this type of study?
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We have done a few experiments where we treat for six months. We seed at a lower density and let the cells grow (in treatment) for 5 days (they are near confluent) then trypsinize cells taking a portion of the cells to continue the treatment with and re-treating. However we have found some strange things happening over the course of 6 months. Looking back we realize that we have selecting for cells with a growth advantage. Problem is 1)this does not likely to reflect actual exposure in vivo and 2) we have possible/probably diluted the affected cells that are of more interest.
Does anyone have any thoughts?
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I have been working with 3d spheroids for a few months. I started with HCT-116 colon cancer cell line and have not had any problems, this cell forms tightly aggregated spheroids. However, I have not been able to form spheroids from HT-29 cell line. I've tried different agarose concentrations (1-3%) and different cell concentrations (800-10.000 per well) and this cell line won't form the spheroids, just cell clumps. Does anyone have any idea what could be happening? I've seen in many papers spheroids from HT-29 and theorically they are formed easily. I use the following protocol for the HCT-116:
96 well plate flat bottom coated with 50uL of 1.5% agarose. I plate 2000 cells and centrifuge 1000rpm for 5 minutes. Then, I incubate for 4 days untill the spheroids are formed.
Thanks in advance.
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Dear Gabriel,
my team and I have recently published a paper on tumor spheroids (https://www.frontiersin.org/articles/10.3389/fimmu.2020.564887/full). We found that HT-29 cells form spheroids with lower weight, diameter and size compared to other CRC cell lines. In the case that you are interested in increasing the sphericity and compactness of HT-29 spheroids I'd suggest using micro patterned ULA plates (Elplasia, AggreWell, SP5D) or adding 1:1 fibroblasts to your 3D cell culture.
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We are located in Toronto, Ontario. I would appreciate your help.
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Hello I am having a bit of an issue with my sf9 cells and trying to express the human PTH receptors. So:
I transform DH10 cells which give me blue/white colonies, which I also do a colony PCR to see if my insert is there and it is. I then proceed to do a bacmid purification (precipitate over night in isopropanol and 70% ethanol just before transfection) and transfect my cells on a 6 well plate. After 72 hours I harvest the media,replace it with 3ml new media and store the baculovirus in the fridge. 72 hours from that I do a western blot which shows my protein was there.
I infected 25ml of sf9 cells with 3ml of my virus on saturday at a density of 1x10(6) they had stopped dividing on monday (48 hours) and then today (72hours) I was going to harvest the virus. However my cells look incredibly stressed with lots of debris, but the media they are in looks perfectly normal, after centrifugation the pellet is normal coloured etc etc. I see no signs of contamination.
Even on the previous 6 well plate my cells looked incredibly stressed with lots of debris, but the media control was normal and insect control looked perfectly fine. I'm assuming I have a contamination that has led to this so I have filtered the virus and will run some more tests but any ideas on what is happening? I'm quite new to insect culture but assumed contaminations would be very apparent? 
Thank you very much for reading!
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Hi, I am also exploring the GPCR expression in Sf9 cells. However, after harvesting the cells, I did not observe any protein expression using the WB. I am wondering which type of buffer you used or is there any matters need attention. Thank you!
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Hello,
I am currently using MRC-5 cell lines and they are at 17th passage. I tried to collect and count them via trypan blue cell counting but I could not see enough cells even though they looked fine in the microscope. I used almost 16 T-25 flasks but couldn't catch enough cells. What could be the problem?
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Christopher L Pinder I am sorry for the late reply. Actually, I realized that they are very adherent and checked the flasks but didn't see a lot. Also, their growth are not bad but started to multiply very slowly. Could they have reached senescence at 17th passage?
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Dear everyone
I need expert's suggestions about my research in Oral Squamous Cell Carcinoma of cell line culture. I will measure the cytokines of P53, BCl2, FasL, and Casp 3 to know the mechanism of apoptosis in this cell line culture after treatment. Please, how to measure using ELISA kits for these cytokines. I am not sure to measure these cytokines from supernatant or cell lysate.
Thank you
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Cytokines expression
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I am establishing a polarized gastric-epithelial monolayer culture on transwell system for bacterial infection studies. I use NCI-N87 cells and I culture them by replacing medium on every alternative day for 21 days. Later, I confirmed the expression of ZO-1 on 100% methanol (-20C) fixed cells. However, I face the following issues during this process.
1. How to avoid membrane curling while I cut off the membrane from the insert to mount on glass slide?
2. When I used 4% formaldehyde as a fixative to stain cell surface proteins, I found few cells or small cell clusters lying over the tight monolayer.
3. Is it necessary to use 21 days grown cells for bacterial infection studies? Because I see many highly cited papers also have used lesser days grown cells.
How to overcome these technicalities?
Any help is highly appreciated.
Thank you in advance.
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Hi Michal Mastarlerz, Thank you for the input. I will definitely enquire about this focus function in our microscope core.
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would like to know how to differentiate them and how are the phenotypic changes? do they give rise to dopaminergic or glutamanergic phenotypes?
what would be the agents to induce differentiation in these cells?
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Hi Abu Bakkar Siddik, I would try differentiation for 48hrs up to 96 hrs even. Also good differentiation markers include Synaptophysin, NeuN, and PSD95
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I am having difficulties in reviving the chicken embryonic fibroblast cell line DF-1. I froze down the cells in complete DMEM medium plus 5% DMSO (half of a entire T75 flask) and thawed following the same procedures as other cell lines. However, even though the cells would attach on the next day, they started to die and float on day 2. I am thinking that the antibiotics (pen-strep) in the culture medium may affect DF-1 growth but shouldn't they only inhibit prokaryotic cell's protein synthesis (as for streptomycin)?? Has anyone come across the same problem?
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Thanks, Guanqun Liu for your reply, I will take it in consideration. my regards.
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Dear all, recently I used a TCR- murine T cell hybridoma 58-/- CD8+cell line to do the calcium flux assay. But Prof. Bernard MALISSEN, the creator of this cell line, told me the cell line can not flux much calcium. So does anyone know a nice TCR- murine T cell hybridoma TCR alpha-/beta- CD8alpha+/beta+ cell line which can get a robust  calcium flux when I use anti-CD3(145-2C11) and anti-IgG to trigger them? I need to transfect BglII/SalI-linearized pBJNeo-TCR alpha and SalI-digested pSH-TCR beta into the cell line and get a stable T cell transfectant.
Thank you very much!
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does anyone know if the 58-/- hybridomas express endogenous CD28?
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Hello everyone,
I am using hanging drop method to make 3D cell culture. But I am struggling with handle the spheres when I exchange the media.
Please help me if you have any experience with this problem. Thank you so much!!!
Best,
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Do the aggregated cells stay in the floating culture afterwards? or they should be separated to prevent growing altogether? Thanks!
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I see this contamination in Caco-2 cell line , cultured in 24 transwells. At the end of growth period of 21 days there is no reduction in TEER values and the control compounds work out well (permeability value ranges are within historical range). Media colour changes to slight yellowish. can anyone suggest which type of contamination is this.
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This debris is under the transwell inserts with cells above, right? I think it is cell debris from the Caco-2 cells, prehaps blebbing-off vesicles to the media below.
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I am working with primary adipose-derived stem cells and have the problem that after plating the cells on 12-well-plates they all move to the center of the wells. I tried several things (8-shaped shaking, different volumes and seeding densitites, incubating the cells on a flat bench for 60 min before putting them into the incubator etc.) which all did not solve the problem. Plating other cells (for example NIH-3T3 cells does not lead to the problem - the distribution is very even so that I assume that it's a cell type-specific problem). Now my hypothesis is that the vibrations of the used incubator is the main issue. Are there any possibilities to somehow catch the vibrations inside the incubator? I found some "anti vibrating pads" in the internet for example. Maybe someone has another idea.
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Have you overcome this problem? I got the same problem when I plated colon cancer cell (HCT-116) in 12-well plate. I tried different shaking method, I put my cell in a flask bench around 15 min before putting in the incubator. I feel the cell evenly distributed, but after 30 min putting in the incubator, I checked again, my cell continues to move in the center.
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It seems that resazurin-based viability assays (such as Alamar Blue) are frequently used with hepatocytes or hepatic cell lines cultures, but the effect of resaruzin on the induction of cytochroms P450 on the samples is barely discussed. As these tests are usually non-destructive, it is appealing to use the same sample for the viability assay then CYP induction analysis, but I can't find an actual proof that resazurin won't affect the CYP activity. Does anyone have feedback or insight on this question? Thanks a lot!
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Hi Timothée,
Incubation with resazurin definitely affects the parameters of cells. Viability reestimated in 24 or 48 hr was lower than in cells treated in the same manner but without resazurin. Moreover, it sensitizes HaCaT cells to following exposure to UV-B.
Taking into account the structure of resazurin it should somehow affect the detoxication system. But I still have no proves.
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Hi everyone,
I would like to add some guanine, cytosine and adenine, independently, to cell culture. I can't dissolve them in DMSO or in water, even turning up the temperature until 95°C. Only cytosine dissolve at 55°C but precipitate again when the temperature goes down. On the specification sheets of the products, it is written they are soluble in HCl.
- HCl 0.5M for cytosine up to 100mM
- HCl 0.5M for adenine up to 20mg/mL
- HCl 5M for guanine up to 25mg/mL
At the concentrations I would like to use them on the cells, even trying to prepare these bases at a 1000X concentration, two problems come to me:
- I'm not even sure they can dissolve in the appropriate volume for the 1000X concentration: the mass/volume being higher than the specifications
- I tried to dilute, even the lowest HCl concentration (0.5M), a thousand time in my cell culture medium and measured the pH with a pH paper --> pH came down to 6. This is not suitable for cell culture.
Do you have any idea how to dissolve these bases?
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Adenine can be dissolved in water by stirring at 60 0C and remains long after the temperature decreased to RT.
Cytosine dissolves in water by stirring at RT.
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Dear experts,
I would like to work on THP-1 as adherent cells without changing its natural. Is there any method or protocol for develop adherence cell from suspension cell lines?
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Hello Dr.Stalin! In our laboratory we promote their adhesion by addition of phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/ml. This conducts the differentation of THP-1 monocytes into macrophages. However, when you say "without changing its natural I do not know if you mean to preserve their monocyte phenotype". THP-1 cells are non-adherent, so all treatments you perform to promote their attachment are going to have an impact on them. For instance, PMA tends to upregulate the expression of some genes in differentiated macrophages, which could affect the gene expression induced by other stimuli. If I am not wrong and I remember well, the treatment enhances inflammatory genes through NFKB pathway. In our case, as we are studying inflammation, we added a period of arrest, leaving the cells without PMA in order to reduce that possible enhanced expression.
I would recommend you (depending on the experiments you have to perform) to try different concentrations of PMA in order to add the minimum necessary to promote their adhesion without enhancing the gene expression. Firstly, I would check the expression of inflammatory genes after the treatment and after the arrest, to see how the treatment affected your cells and whether the arrest decreased the inflammatory response. Lastly, I would characterize the cells through flow cytometry or gene expression of monocyte/macrophage markers to see the profile of your cells.
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Regarding price issue, I want to use NBCS instead of FBS for the culture of THP1 cell lines. If any body has experience (good/bad), please share it.
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Thank you everyone.
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I need to culture mammalian cells on a 60mm culture dish in an airtight container with 5% CO2 gas (5mL growth medium).
Are there any rules / published data on the ratio of medium : atmosphere that is required for optimum cell growth in closed environments? E.g. 1:20 ratio maintains stable gas concentrations for 24 hours.
Also, are there any publications describing the effects of closed culture on mammalian cells?
Thanks!
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I have expierence with closed culture system only for delivery culture cells, I help a coworker that need to transport the lived cells. Then I used the system for some hypoxia study. maybe you can find some information about required volume of gas on hypoxia article.
Regarding study effects of closed systems I think that you can find something on web i don't understand if you need this on primary cells, for clinical purpose or something else.
For example, I can't download the article but you search something like this?
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So recently I started working with HepG2 cell line and I'm having the hardest time to evaluate their confluency due to their morphology and the formation of aggregates, so I was wondering if anyone can tell me what's the confluency of the hepG2 in the photo and if I should sub-cultivate them in this state.
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I think this looks not like HepG2 cell ,not epithelial in morphology but looks like frbroblast cell
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My 293t cell morphology is strange. it has a lot of pseudopodia and its like a spider web. I thawed the cell just recently. And my culture media is DMEM+10%FBS+antibiotics.
Has anyone encountered the same problem? What can cause this problem?
THANKS
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They look pretty similar to these HEK293 at Elabscience, just more dense: https://www.elabscience.com/p-293_[hek_293]_cell_line-58964.html
Also pretty similar to this lot of HEK293T at ATCC: https://www.atcc.org/products/all/CRL-3216.aspx#characteristics
I wound be interesting to see how they look at different densities during growth.
Different batches of HEK293/HEK293T cells can have slightly different morphology and tendensy to grow as islands. According to the literature, the morphology and the strength of attachment can vary with different lots of FBS. The density after dilution and detachment/attachment propensity will also affect the final patterns at high density.
The batch of HEK293T (tsA201) I am using is less clumped together at lower density but eventually they all can form a sense layer.
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Hei,
I want to analyze apoptosis using AnnexinV/ PI. I am working with various neuroblastoma cell lines such as SKNAS, SHSY5Y SKNBE(2), Kelly and several other.
I am using the FITC-AnnexinV/PI kit from BD.
The assay worked always fine when I analyzed apoptosis in SKNAS.
However, when I used the same protocol for SKNBE(2), I always got approximately 80% Annexin potitive cells. And these cells were not treated- thus, these cells were healthy cells that should not have more than 5-10% apoptotic cells.
Today, I analyzed apoptosis of Kelly and SHSY5Y cells. Here, I also got 70-80% Annexin positive cells in untreated cells.
Might the cell membrane of these cell lines have phosphatidylserine in the outer leaflet of the plasma membrane even if cells are not apoptotic?
If so, the assay would not work for these cell lines...Did you have similar problems when using this assay or read about it?
This is my protocol:
  1. Transfer medium to 1.5 ml tubes
  2. Wash with 300 μl PBS and transfer PBS to 1.5 ml tubes
  3. Add 200 μl trypsin and incubate 3 minutes
  4. Use the medium to inactivate trypsin
  5. Transfer the cell suspension back to 1,5 ml tubes
  6. Centrifuge at 200 g 5 minutes
  7. Resuspend cells in 500 μl PBS (2 wells are merged)
  8. Centrifuge at 200 g 5 minutes
  9. Resuspend in 100 μl Annexin V binding buffer
  10. Add 5μl FITC-Annexin V and PI
  11. Gently vortex cells and incubate 15 minutes at RT in the dark
  12. Add 400 μl 1x binding buffer to each tube
  13. Analyze by flow cytometry
PI:  Laser 561 nm; Filter 670/30
AnnexinV: Laser 488nm; Filter 530/30
Controls:
  • unstained cells (to set gates)
  • PI only
  • AnnexinV only
  • PI/AnnexinV
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Hi Sarah,
I am also facing the same problem with Neuro 2A (Mouse Neuroblastoma) cell line. In healthy cell, I am also getting 60-70% cell viability. To troubleshoot this, initially, I ran the cell with 10, 15 and 20 % FBS and got 70% (Max) cell viability in 15% FBS Cells. Then after used gradient of PI (3, 5 and 10 μl ) and annexin but still got the same result. Kindly suggest to me If you have figured out this problem. Thank you in advance
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Dear colleagues, currently I am doing an inflammasome-related experiment using cells. I have a substance as a candidate for anti-inflammasome. However, I am a little bit confused about which step I should administrate my candidate regarding the inflammasome molecular pathway. Is it better after primary inducer (LPS) and before secondary inducer (ATP) or simultaneously administrate with the secondary inducer (ATP)? I beg your suggestion and further consideration. Thank you.
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I agree with the previous comments. I would like to add that if the potency of the "substance" as an anti-NLRP3 inflammasome activation is not known, optimization of treatment may be needed- treat before "priming" and after the "priming" assuming that the "substance" does not alter the levels of NLRP3, ASC, and pro-caspase-1 in your cells.
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The breast cancer lines MCF-7 will be cultured, stimulated and then a pathogenic bacteria will be introduced, incubated and screened for cytotoxicity, but I am kind of exploring these cell lines regarding which cytokines are expressed in case of induced infection? Or is it just specific to the bacteria that I will use for the study and the induction method?
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Check CCLE RNAseq data
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Hello everyone,
I am trying to bring a line cell of a mammary gland from Brazil to Chile. From the University of Sao Paulo, to be more specific. However, in order to bring them cryopreserved, they must travel in dry ice, a product that is difficult to pass through both borders.
That is why I am trying to see the possibility of bringing them in T75 flask with a lot of culture medium through DHL. So I am looking for someone from Brazil, hopefully from the University of Sao Paulo, that can offer me the services of cultivating my cell line and then to send me Flask T75 from Brazil to Chile. The idea is that if the cells arrive to Chile dead for some reason, there is backup cells in the laboratory in Brazil. Therefore, the service I need, would be to grow cells and to make a backup of these cells to keep in their lab. These cells do not require anything special. To grow these cells you will only need to use DMEM medium with 10% FBS and 1% antibiotics in an oven with 5% CO2 and at 37 ° C - 38.5 ° C.
We will run with all the costs. At the moment my cells are in a laboratory of the University of Sao Paulo and I need to be able to bring them urgently. This because I need them for my doctoral thesis. I already tried to bring them through World Courier but it is extremely expensive to do so.
Anyone who is able to help me please contact me through my email: e.munoz09@ufromail.cl
Thank you for your attention!
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hi
By an air traveler, you can transfer the cell to your laboratory immediately after defrizzing and placed in a 15 ml falcon's tube include complete culture medium for 12 to 24 hours.
good luck
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I am using 10% FBS RPMI medium with 1% Sodium Pyruvate, penicillin streptomycin and MEM non-essential amino acids. If I use 20% FBS the cells become giant vacules rather then proliferating. I split them every 6-7 days. They are nearly impossible to work on or do an experiment with this proliferation rate. What can I do?
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I had similar problem with SHSY5Y neuroblastoma cell-line. Later I figured out two possible reasons:
1.pH of media used
2.quality of FBS used in the culture medium. You can try to change these and see if the cell grows.
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Hi,
i am working on effects of gemcitabine in HepG2 cell line and i should use the IC50 of Gemcitabine in the experiments.i found different values in literature and the numbers are highly variable ranging from lower nanomolar to lower micromolar for the same cell line under same conditions.!!!!!!
the IC50 that i got from my experiments are different from loterature and also is variable in each test of mine.....
in each test i got different IC50 and the conditions are the same in all experiments.what should i do?
the source of gemcitabine that i use is powder and the solution of gemcitabine is unstable and each time i should make it...
do you have any suggestion to solve this problem?
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Mr Michael Theobald thanks for article that you suggest. and the expiry date on the drug package is for year 2020.
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Hi everybody,
I'm treating my contaminated cell cultures by primocin. What experiences do you have? What is the optimal length of treatment to obtain healthy cell culture? Do you use primocin all the time as preventive step or only in the case of contamination? 
Thanks a lot
Pavla
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Routine use of antibiotics in cell culture is not recommended. since , antibiotic resistant strains may develop and may cause resistant cryptic infections such as mycoplasma. Moreover, some antibiotics may have effects in cellular functions. Otherwise, primocin is a good choice.
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Does anybody has any experience of fibroblast cell line culture without employing a CO2 incubator?
Thank you very much
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The reason that mammalian cells are usually cultivated in a 95% air/5% CO2 environment is to maintain (i.e. buffer) the pH of the media via the HCO3 ion. Other buffering systems are available and do work. @Ron has identified one that is popular: HEPES. I suggest that you read the technical notes from Corning, Lonza, Invitrogen, even Sigma (now Merck).
FYI, this question was asked, and answered, previously in 2013: www.researchgate.net/post/No_Co2_incubator-can_anyone_help.
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Dear all,
I am planning to stably transfect a cell line with a luciferase reporter gene and an antibiotic selection gene (neomycine).
Now I'm doubting whether I should remove the neomycine selection cassette or not (using flox/cre) after I have selected my clones. Do you feel this is necessary?
Once selected, I don't want to keep them in culture with neomycine. Is it lethal for cells to stop adding neomycine when they have the neo res cassette or are there other considerations I should keep in mind?
If you need any more info, do please ask.
Thanks in advance
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Thanks for all the tips! This was extremely helpful
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Hi,
for my research I need to use laminin for some patterning and am wondering whether the C17.2 cells (neural progenitor cell line) be cultured on it (instead of poly-l-lysine as suggested by the distributor and papers).
The first test schows that they do attach without problems, but is there a risk that they will they de-differentiate?
Would be grateful for any experience with these cells or other neural progenitor cells regarding the coating!
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The cells will certainly grow on laminin but it will change their biology. The answer depends on what experiments you are planning. I would stay with the suggested approach.
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I want to study some ischemic reperfusion effects on heart.
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Dear Colleague you can buy them from cell bank of pasteur institute of Iran too.with bests
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Hi,
Our B16F10 cell line from ATCC encountered massive cell death after switching the growth media from RPMI to DMEM.
We were stuck with RPMI( 10% FBS, 5ml P/S) for the first 2 passages with the lab mistakenly ordered DMEM w/o sodium pyruvate & l-glutamine. The B16F10 cells looked healthy as we split them at the subcultivation rate of 1:5 and passaged them every 3-4 days.
Then, the growth media was replaced with DMEM when the glutiMAX arrived. 5mL of glutamax (5mM) + 10% FBS, 5mL p/s was added to prepare the DMEM. The cells were split 4 days ago with the new DMEM and at a much lower ratio of 1:12. We checked the cells today and they were all dead. Shown in the pictures.
Any advice for the troubleshoot would be very much appreciated! Thank you in advice for the help!
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B16 variants require media with specific AAs to form melanin. Thus, in RPMI-1640 they loose the brown color. It is best to culture in the recommended media of MEM with additional NEAA and EAA. This maintains the melanin.
As mentioned B16 variants are adherent cells, but when they become acidic or nutrient deprived they form cell mats and also release cells from the plastic surface. Further, they form exosomes and appear to have a large number of floating cell fragments. Thus, a 2 to 3 time per week split is optimal. Also assure that the split results in about 20% confluence.
Hope this helps.