Cell Line Culture - Science method

I am preparing to work with Caki-1 and Caki-2 cell line cultures. I've seen a couple of biotech company websites suggesting the inclusion of 2 mM Glutamine to the McCoy's Modified Media that these cells require. Some other websites have no mention of the Glutamine addition so I want to know if Glutamine is necessary. Any advice is appreciated!

I recently purchased cryopreserved SW-982 cells. I thawed them according to the manufacturer’s protocol and cultured them in RPMI medium supplemented with 10% FBS, 1% antibiotics, and 1% glutamine (I’m unable to use L15 medium as my incubator is set to 5% CO2). From the start, the cells have been growing in suspension, forming spheres, with very few adhering to the flask surface.

I contacted the company, but they were unable to identify the cause of the issue.

Has anyone encountered this before? Why are they growing in this way, and what can I do to make them grow forming a monolayer?

I had mammalian cancer cell line cultured and fixed for confocal imaging. While observing I found these particles around cell nuclei stained with dapi. I checked the main culture for bacteria but there was no bacterial contamination. Are these mycoplasma?

I am currently working on iPSCs and we usually culture them in mTSER+ media. In our routine mycoplasma testing, we found that all cell lines cultured in the mTESR+ media were positive for mycoplasma, and after some investigation, identified the source was the mTESR+ 5X supplements (Stem Cell Technologies : Cat#100-0275, Lot#1000146377). Freshly thawed and opened supplements independently tested positive on two separate occasions, with two different sets of mycoplasma primers. The basal media tested negative. I was wondering if anyone else faced this issue or have product from the same lot who can verify their products as well.

I have purchased HK-2 cell line from ATCC and using Keratinocyte serum free medium plus 0.05mg/ml BPE plus 5ng/ml EGF purchased from invitrogen. Actually, the main problem what I am facing is that after 5th day when I was ready for splitting, they become rounded off and dies suddenly. On 4th day at 4 pm i have seen that cells were fine . But entire scenario is changed at morning. The media pH and color was normal. suddenly what happened to cells that am not able to clear it. If you have any idea kindly let me know. it will b great help. Thanks

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

Hi everyone,

I am culturing bone marrow derived stem cell now and trying to do osteogenic differentiation. I found a lot of black dots after the cells get confluence which can be washed away with PBS, but came back again at the next day and  there are much more dots in expansion medium compared with osteogenic media.

1. These dots just vibrate at almost the same place, do not move actively. After changing media two days, the expansion media looks little yellowish and osteogenic medium seems okay.

2. We can not see the medium become cloudy and cells in flask always looks fine without any visible dots (less than 80% confluent).

3. Our expansion media is alpha-MEM + 10% FBS + 1 ng/ml FGF-b.

Can anyone tell what are these things or they are just something secreted by cells? I did not notice this phenomenon before the end of last year and it's totally the same cell and same medium. 

Cells 1-3 were in expansion medium, 4-5 were in osteogenic medium with different magnifications. 

HI

i transfected adenovector to hek293 cell line. i don't know Adenovirus CPE.

this picture is hek 293 cell line 8 days post transfection. do you observe any CPE?

I am maintaining MCF7 in 10% FBS and 1% Penicillin/Streptomycin DMEM medium and there was a formation of aggregates after passaging by 3 days. The growth medium color wasn't changed. Could you please help me identify this contamination? The pictures were attached below.

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.

Hi!

I am staining iNOS and CD163 in my cells so I am looking for a minus control, expressing neither iNOS nor CD163. But the cell familiar in our lab including panc-1 and 293T seems at least expressing 1 of them. Which cell line is known to not having observable level of these 2 proteins?

Thanks!

Has anyone had any success with Crispr and MDA-MB-231 or MCF-7 cell lines?

I have been experiencing some trouble culturing Hep3B cells since a while now. I use DMEM with 10% FBS and PenStrep. On reviving a vial the cells grow very slow and even if they start growing well, they suddenly die and the growth is stopped altogether. 

I am referring here predominantly to therapeutic CHO cell lines, although this trend does seem to be widespread. In the literature, often in the same study, electroporation is used to generate stable cell lines, whereas a chemical method such as PEI-mediated transfection is utilised for transient gene expression. This is true for industry and academia as far as i can tell. Reviews on mammalian transfection methodologies tend to argue that chemical methods are by far the most common, for a list of reasons that make it more advantageous. Does anyone know of any reason why people continue with electroporation for stable work? If I was to guess I would say that it is more efficient at DNA delivery and that the hit taken in cell viability is not so important, because stable cell line generation allows plenty of time for recovery and perhaps also because regulatory bodies might not be comfortable with potential lingering chemicals in formulated products. However, I cannot find any literature to support this. Any help would be greatly appreciated. 

Thanks in advance,

Joe

I cultured the Hela cell line for anticancer testing of a plant extract. On the 3rd day of subculture and changing the media, the cell culture showed that the DMEM medium was cloudy and looked like small particles when observed with an inverted microscope at 40X (as seen in the attached picture). What do you think contaminated the HeLa culture cells? Has anyone experienced the same as me?

Hello all,

I have recently embarked into mammalian cell culture using mutiwell culture plates (e.g. 6-well plates, 12-well plates, etc.) as opposed to previous studies where I have used individual flasks (e.g. T25, T75, etc.). When using flasks, I was accustomed to lysing cells in somewhat of a more "low-throughput" method in a lysis buffer containing 1% Triton X-100, applying mechanical shearing force by passing the cells through an 18G needle.

Moving to multiwell culture plates, the needle method is quite tedious. Having to pass the cells from each well through an 18G needle, one at a time, is very time consuming and counterbalances much of the time that is saved by using a multiwell plate in the first place.

Is there another method that is friendlier to "high-throughput" multiwell plates, that anyone might suggest which lyses cells without having to pass them through a needle one-by-one -- yet does not interfere with downstream assays for protein concentration?

Thanks in advance,

Chris Dieni

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.

Does anyone is culturing the Wehi-3 cell line? We got it from ATCC and following the ATCC guidelines but cells are not dividing/ numbers not increasing.

Can anyone suggest

Do I need to add osteogenic induction media to hFOB1.19 (CRL-11372) cell line to hFOB1.19 (CRL-11372) cell line if I want the cells to differentiate to mature osteoblast? or these cells are osteoblast already? This is my first time hOB cell line culture. Some studies incubate at temperature 39.5°C for differentiating temperature. Is it necessary? or is it OK with Growth Conditions at temp of 34°C? ( recommended by manufacturer )

- If osteogenic media need to be added the media would contains Ham's F12 Medium Dulbecco's Modified Eagle's Medium, with 2.5 mM L-glutamine (without phenol red), 0.3 mg/ml G418, 10% fetal bovine serum supplemented with 0.1 µM dexamethasone, 50 μM ascorbic acid and 10 mM β-glycerol phosphate

Thank you

Hello,

I have been trying to passage ma-mel-66a cell line, however, I am having zero luck. I have used RPMI + 10%FBS and RPMI + 5%FBS, I even plated them (straight from LN2) in a small p10 to help them out. Nope, nodda, nothing. Can anyone recommend me a useful protocol or media formulation for this cell line?

Thanks!

J

Dear all,

I am starting working with E0771 cell line since I have to establish an orthotopic breast tumor model in c57 mice but I have no experience with this cell line so I would really appreciate any advice that you can give me.

In particular, I saw the ATCC website and they say that it is better to culture these cells in a t-75 corning flask, maintaining cultures at a cell concentration between 6 x 10^4 and 8 x 10^4 cells/cm2, is this true also for your experience? How many cells do you plate in a 75 cm2 flask?

Do they grow fast? How many times per week do you subculture them?

Sorry for all of these questions but I am new with these cells and so I would really be very grateful for all your advice,

Thanks a lot,

Giulia

I have experienced this problem previously with a commercial cell line, however in this case, a well established immortalized cell line when cultured from frozen stock has cell populations that have a bright border around them. This has never been the issue with this particular immortalized cell line. I am wondering if there could be possible mycoplasma contamination. Any comments on this are highly appreciated. Thanks!

Hi!

I need to prove that my protein is localized on the cell-surface but not inside the cell in soluble form.

How can I make it?

There is an opinion to wash cells, to label all cell-surface proteins with NHS-biotin, and to collect labelled fraction of cell-surface proteins with streptavidin magnetic beads. After that I can make western blot with specific Ab.

Maybe there is another way?

Protein and cell lines are human.

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.

Does anyone have experience with CAEV in immortalized cell lines? Some publications show use of goat synovial membrane cells or goat milk epithelial cells, but these cell lines are not available through commercial companies.

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?

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!

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

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.

Hi Everyone,

Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?

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.

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.

What is the time taken to reach confluence for hepg2, huh7, heap 16 cell lines cultured in 10%FBS, DMEM high glucose

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?

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.

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?

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.

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?

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.

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. 

deleted

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?

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.

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

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!

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!

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?

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.

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.

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

I want to do some cell migration assays and I would like to know if it's possible to grow these cells in suspension

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 ?

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.

I am passaging HEK 293 cells for retroviral transfection.

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?

Hi, I had a lot of problems with this cell line. When I count the number of cells, I can't do it, because the cells form a lot of cell aggregations and i can't separate them. The cells was incubated with trypsin for 2 minutes, and the cells were detached of plate, but the cells form aggregations. So, i have a lot of problems with the MTT assay due to the number of cells is not very real. If somebody can help me... I dont' know if the problem could be the medium, because i was used DMEM instead of McCoy...and i don't know if could be this the problem.

Thanks!!

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?

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

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.

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?

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!

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!

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. 

I am working with raw cell line from last one year and they were fine before .But since last month they keep on self activating.. ( without treating any activating agent like LPS) even after thawing during 1st passage their morphology changes completely from round to star shape. 

I have tried using different stocks...

All other cell lines in our incubator are fine so I think its not incubator problem, even then i tried to clean it all over again but all in vain..

what should I try .. ??

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?

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?

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.