Cell Culture Techniques - Science method

Hello all. I grew some cells for virus infection. I infected the cells with CMV (Cytomegalovirus) 8 days ago. The cells have not started lysis yet, but the plate is about to get dry. I wonder if I can add 5 mL of growth medium (DMEM) to the plate containing CMV-infected cells to prevent dryness at this moment (8 days after infection). Is there any risk to do so? Any advice is appreciated.

Hello all. I grew ARPE-19 cells in cell culture and infected them with a virus (Varicella-zoster virus). After the virus reached a high infection rate, I harvested everything in the plate and use freeze & thaw technique to release viruses into the supernatant. Now I want to store my viruses in -80 freezer. What is the composition of freezing medium for VZV? Are DMSO and FBS enough? Or do I need to add sucrose or something else? This is a bit urgent. I have no one to ask because I am the last employee in my lab as my professor is retiring. Any advice is appreciated.

Hi everyone,

I'm interested in finding a protocol for detaching neurons in culture for use in flow cytometry. The main issue I can foresee is in the production of a single cell suspension. Any help would be greatly appreciated.

Tom  

Hello everyone,

I am facing a frustrating issue with the TC28a2 cell line. After treatment with staurosporine at concentrations of 25nM, 50nM, and 100nM, the cells begin detaching during the first media aspiration when I switch to fresh complete media. This problem occurs regardless of the incubation time. Does anyone have any advice on how to aspirate the media without losing cells?

I'm just wondering if I need to change the media of my cells that are culturing in 5% O2 (hypoxic condition) in a hypoxic chamber aka a hypoxic glove box? Or can I just quickly do this in a normal incubator and return the cells to the hypoxic incubator?

It sounds like a lot of work if we need to get a hypoxic glove box so I'm just wondering if anyone has any experience in this who could give me some insight?

Thank you

Dear all,

I prepared my cell media (EMEM+10%FBS) and another cell media (EMEM+2%FBS+1% Pen/Strep). Incubate the aliquot of the media in 37°C for a few days and saw these. They are not moving and there was no color change in the media. Since there were no cells in the flask so if these weren't cell debris, I wonder if this is debris/aggregates from the serum? I also have done sterility check on the serum and no changes observed to indicate contamination.

Thanks.

Hi everyone,

I have been having some issues with SHSY5Y cells that I never had in the past. I used to culture them in DMEM:F12 with 10% FBS and 1%Antibiotic-Antimycotic and they grew just fine. Back in October, when we received a new batch of medium, I started seeing increased cell death after 1-2 passages: specifically, I would see debris like structures and the cells literally peeling off from the bottom of the flask. I initially attributed this to the quality of the medium and ruled out incubator, FBS etc issues. I thawed a new vial of cells and I had the same issue. Then I was told to try OptiMEM and with a new vial from ATCC I noticed that they grew beautifully and reached confluence within 3 days. After the 3rd passage, I started seeing the same type of pattern that I saw before--cells fragmenting, a lot of debris and cells peeling off and floating in suspension 1-2 days after plating. I am desperate as my lab has wasted many cell stocks and money on trying to figure out what this is. My next step is to test for mycoplasma but I wasn't sure if this is the case considering that we literally just bought a new stock from ATCC and it grew fine until the third passage.The medium doesn't yellow out and it is not yeast contamination. What can be the source of this type of behavior?Any input is greatly appreciated! :)

In the picture derived from light microscopy, there are some red stains apart from the blue ones. I am wondering, what could it be?

After mature adipocytes isolation from human adipose tissue, I want to adhere them for my studies (Adipocytes tend to suspend on top layer of the media). Which method or materials do you recommend for coating (with a minimum effects of adipocytes differentiation, shapes and viability)? Poly-L-Lysin? Collagen-coating? etc. Thank you so much for your guidance.

Hello everyone,

I renovate the cell list for the cells we store in -196 liquid nitrogen tank in our lab and I wonder what you include in your list for this. Cell name, passage number and the date I passage and store them in the tank are my elements for the list. What would you add beside these?

Thank you.

I am trying to revive my cells and I don't know what they're being affected by. I have thoroughly cleaned every corner of the culture room and incubator; done all possible cleaning practices to avoid all possible sources of contamination. Now my cells are not adhering to the surfaces of the plates. They become rounded and are floating in the media. What additional techniques can I try to establish my culture? I don't know whether it is some biological contamination or some physical factor that is playing a major role.

I am currently attempting to culture cell lines in a high %CO₂ incubator to mimic hypoxic conditions. Unfortunately we do not have incubators that can adjust the O₂, nor do I have access to a hypoxic chamber so increasing the CO₂ to 20% seems to be my only option.

The resulting issue: cell culture media typically contains a sodium bicarbonate buffering system that is optimised for incubators set between 5-10% CO₂, so in a 20% CO₂ incubator the media becomes slightly acidic.

Theoretically, I could increase in concentration of NaHCO₃ to 8g/L for 20% CO₂ to buffer the media to a pH of 7.4 (a reference for the calculation used to obtain this value https://www.researchgate.net/deref/https%3A%2F%2Ftools.thermofisher.com%2Fcontent%2Fsfs%2Fbrochures%2FD19558.pdf?_tp=eyJjb250ZXh0Ijp7ImZpcnN0UGFnZSI6InNpZ251cCIsInBhZ2UiOiJxdWVzdGlvbiIsInBvc2l0aW9uIjoicGFnZUNvbnRlbnQifX0), this however leads to changes in the osmolarity that my cell lines can't seem to handle.

Does anyone have any suggestions on how I could adjust my cell culture media to suitably culture cells in 20%CO₂?

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

Hello everyone,

I'm curently working on HaCaT cells which require low calcium medium to return to undifferentiate state. To do so, I need to chelate my FBS for 1h with chelex resin among other things.

Typical protocol suggests to chelate FBS, add to medium, then sterile filter the complete medium.

As only the FBS + chelex is not sterile and because filters are not that cheap, I rather filter sterilize a complete bottle of chelated FBS and then use it to complete my medium bottle.

Problem is the resin is clogging my 0.22µm filter, even 0.45µm get clogged before getting the 500mL of FBS passed through...

I was wondering if anyone had come across this problem and may have a handy solution.

- should I centrifuge the bottle before filtration and leave a dead volume ?

(i tried 5min 1500g but it didn't help much) maybe go higher ?

- maybe pass the FBS through a 80µm cellular sieve ?

- any ideas ?

Thanks for your time,

Philippe.

Hello, is anyone doing cell culture for shrimp hemocytes? I have a problem with cell sorting; I cannot get the cell sorted out. I hypothesize that I used the L15 medium and did not adjust the osmolarity. Does anyone know about the L15 medium? Should we adjust the osmolarity of the L15 medium based on the osmolarity of shrimp hemocytes?

In some studies (especially cancer research), a relevant cell line (e.g. endometrial cell line) is used in cell culture together with an unrelated cell line (e.g. kidney cell line). However, the unrelated cell line is not used in all subsequent experiments. What is the reason for this?

Do I have to use different cell lines in my study?

What are the validation timelines for quantative real time PCR and western blotting results, is it 24 hours for qpcr and 48 hours for western blotting if so! Why then!?

I've been growing these cells for a while, but they're not growing as fast as they should, and they're look weak and I've Spheral shapes round cells, does anyone know what these are?

I am trying to test the cytotoxicity of my zein scaffolds by direct contact assay, but the problem that the scaffolds are floating in the medium, so the upper surface is not covered by the medium, so I can't seed the cells.

Any solution for this problem.

P.S. I need to test the cytotoxicity through direct contact, not using the scaffold extract.  Thanks in advance.

I am doing a colony formation assay on 96 well plates. Each well has 150 NTERA-D2 cells. The incubation times for this assay are 4 days, 7 days, 10 days, and 14 days.

I replenish the media every 4 days. However, on day 8 I noticed that when I replenished the media in the untreated negative controls, some of the cells in the center of the colony had detached or died (please refer to the photos to know exactly what I mean). When I observed the cells again on day 10, the cells in the control looked dead (shrunken and spherical).

I am very gentle when I replace the media in the wells, so I do not know why they are getting detached or dying. Is there anything I could do to stop my cells from dying?

The passage number of the cells was 18.

Using a 24-well plate or a 12-well plate is not an option either.

I have attached a photo of what healthy colonies should look like as a reference.

Any help is greatly appreciated.

Urgent question! Do human cells survive at -20 degrees for couple of weeks or do we have to be store them at -80 or below. Any suggestion will be greatly appreciated?

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

We are culturing hESCs using TeSR AOF kit and passaging with Accutase. The cells were in good condition, but one day after passaging the colonies suddenly formed weird shapes and every day there are a lot of cells dying.

It seems cells are being excluded from the colonies and died. Has anyone encountered such problems?

Hi,

I am carrying out the MTT assay using BJ-5ta fibroblasts cell line to evaluate the cytotoxicity of the nanocomposite hydrogels. I want guidance about deciding the controls in the experiment.

blank: media+MTT solution (to cancel the background noise)

positive control: Cells+media+MTT solution

control hydrogel (containing no nanoparticles)

hydrogels (containing different concentrations of the nanoparticles)

negative control : here I am confused and need guidance, whether it should be DMSO+cells+MTT solution

I will be grateful for your help!

I want to check the cytotoxicity of my material. I am using MTT assay kit. The protocol is provided in the booklet with kit how to perform the assay. Is it better to follow the protocol provided in the kit or use protocol given in literature review. As in literature review different protocols are given performed with different kits.

I culture human iPSC in monolayer for my research, kept in 6 well plates coated with either matrigel or laminin, and kept in either mtesr or iPSC brew (sometimes a 1:1 mixture). Previously they have always passaged fine. Recently the very same line doesn’t passage well because when centrifuged, they don’t form a cell pellet. I am lifting them using 3 minutes of relesr in the incubator followed by scraping. They lift fine; this isn’t the issue. But when I centrifuge them at 300g for 5m (what I’ve always used) they remain suspended and do not form a pellet. I even will centrifuge again at up to 800g for 6 min - still no pellet. Any ideas why?! Please assist!

I’ve queries regarding cryopreservation of mammalian cells in DMSO the queries are as fallows:

Q) How DMSO will protect cells from intracellular ice formation during freezing?

What I know is It will penetrate into the cells the push out the water from cells so that no ice crystals are formed , If this is the case then

Q) During freezing DMSO will penetrate inside the cells and displace the water out from the cells thereby preventing ice crystal formation inside the cells, But the water that was displaced out from the cell will form ice crystals during freezing, So won’t they cause any damage to cells (i.e damage caused due to ice formation by extracellular water)?

Q) DMSO is toxic to cells, But during freezing the DMSO will enter inside the cells in that case won’t it be toxic to cells (I mean DMSO outside is toxic but inside is not toxic to cells)?

Q) During freezing DMSO will penetrate inside the cells and displace the water out from the cells, Whereas during Thawing water penetrate inside and DMSO comes out of the cells why (or) what is the mechanism?

Q) During freezing is there any residual DMSO that was left outside the cell (or) entire DMSO will penetrate into the cells? (I mean During cryo preservation will the entire DMSO penetrate into the cells (or) only some portion will enter and some other portion will remain outside the cell)?

Q) Why DMSO enters into cell during freezing and why it comes out of the cell during thawing?

Q) During Thawing why the penetrated DMSO into the cells will come out? Why can’t it remain inside the cell?

Q) During thawing is there any residual DMSO that was left inside the cell?

Q) During cryopreservation at low temp all the metabolic activities get’s stopped, Then why we have to include Complete Media/ 95% FBS along with cryoprotectant (Such as DMSO), Why can’t we simply cryo preserve in DMSO?

Thank You.

Hello Everyone

I am using 0.25% tryspin to digest my cells for passage/seeding. However, I just notice that the cell quality is not good based on appearance (e.g. many rough cells and clusters are observed). I am troubleshooting my parameters, and I am investigating if trypsinization contributes to this kind of cell quality.

I am planning to reduce my trypsin concentration 10x (so I will use 0.025% trypsin). I talked to my colleagues, and they use lower concentrations of trypsin.

Do you mind if you can share references that support that trypsin concentration affects cell quality?

Thanks.

I am working on a project looking at the cytotoxic effects of lipid oxidation products on caco-2 cells. I would like to include a sample group with added antioxidants as a 'negative control'. What commercially available antioxidants might be suitable or have been used before? What concentrations are generally used? Are there other considerations to keep in mind? Thanks!

0.1 million cells in 1 ml media were added per well. Same pattern observed in all 12 wells during multiple platings.

If so, how expired were your components and how did your results turn out?

I'm working with a suspension CHO cell line and have transfected a plasmid containing zeocin resistance. I'm trying to do the selection to get to a stable population. My cells normally are shaken in suspension. I currently have several flasks in both suspension and T-75 flat flasks in tandem with 2 x 6 well plates for generating a kill curve. 

I've been at it for about 4 weeks and using 700 ug/mL based on the initial data I got from the kill curve. I haven't killed off my non-transfected cells still and I'm getting very different viabilities in the shake flasks vs the T-75 flasks. 

Basically, I am hoping someone has some experience with zeocin or other antibiotic selection in suspension cells. All data I find is in adherent cell lines which makes it a little difficult to know what I'm doing incorrectly or what I should be expecting.

Any help is greatly appreciated! Thanks!

I have 3 nanoparticles and I want to screen it for in vitro wound healing activity on HaCat Cell lines. My lab doesn't has the facility for cell culture techniques and hence, I am looking for any lab in Delhi that could help me in doing this activity. Kindly help me in this regard.

Hi there. Anyone has protocol for TaKaRa PrimeSTAR mutagenesis basal kit in English? I have tried to search online but I can only get a Japanese version.

I am going to work about the cytotoxicity of Cisplatin with cell culture in A2780 and Ov-car cell lines. What is the IC50 value of Cisplatin?

Hey all

I recently revived a vial of adherent oral cancer cells and plated them into a T-75 flask. I could notice so many dead/non-adherent cells floating in the media. I thought this would stop after two subcultures, and I would get a homogenous cell in the same cell cycle phase. However, that is not the scenario. I see so many dead/nonadherent cells.

Kindly help

I've some queries regarding assigning passage no in cell culture

During reviving : The passage no of vial = passage no of culture flask that was used for reviving.

During Freezing : The passage no of Cryopreserved vial is +1 to the passage no of flask that was used for freezing ?

4. For suspension culture :

During reviving : The passage no of vial = passage no of culture flask that was used for reviving.

During Freezing : The passage no of flask that was used for freezing = The passage no of vial ?

Hi, I am using PBS, donkey serum and Tween for blocking solution. Unfortunately , the next day there is fungal contamination in the solution. How can I avoid this contamination for ICC

I have done two tests to investigate the effect of a material to cell viability and how cytotoxic the material is.

Briefly, I seeded cells in a 96-well plate and introduce the cells with the media treated with the material, and then place them in the incubator for 24h before testing. The next day, I did the PrestoBlue test and followed by the LDH assay.

When I did the PrestoBlue, the control contains of huge number of cells which is making sense for me as it is a control. But when I did the LDH assay, the reading between control (media + cells only) is quite high compared to the blank (media only). From what I understand, this means that cells are dying in the media, but this result does not correspond with the PrestoBlue result.

Can anyone help me explain what is going on? Is it a user fault (my fault) or is there any scientific reason why the results are not corresponding to each other?

I am currently trying to grow SK-N-SH cells and they are growing extremely slowly, much slower than any other cell line I have ever used.

I have been trying different formulations of media and am currently using DMEM with 10% FCS and pen/strep.

Does anybody use these cells and get good growth from them with maybe another formulation?

I am dealing with human epithelial cancer lines cultured in T-75 flasks. After trypsinisation, trypsin inactivation and centrifugation, the cell pellets collected were resuspended 30-40 times with fresh media before subculture/seeding. Nevertheless, clumps of cells can be observed along with single cells.

Kindly advice to avoid the cell clumps.

1) Any better angles for the tubes containing pellet and pipette during resuspension?

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

I have been culturing 293T cells since last 2 years and never had this issue. They develop multiple vesicles seeding after 2-3 days, fail to grow and degenerate. I have tried changing media with fresh cells, but with same results. Are they infected or some other deficiency ? Kindly refer to the attached pics. 

Hi,

I am currently working on my master thesis and I want to do a transwell migration assay with bone marrow derived macrophages in January. I have aliquots of the macrophages in liquid nitrogen storage and when I use them for different experiments, I usually let them recover for about 2 days with daily medium changes. The harvest (I scrape them off the dish) and the freezing-thawing process puts a lot of stress on them, so I like to give them this time of recovery and get rid of dead cells.

When I searched for protocols for the migration assay, I only find that the cells are grown in another dish prior to migration and then detached by Trypsin and seeded into the transwells for the migration. I would like to go around this detachment-step, as it would be additional stress for the macrophages. So could I also seed the macrophages directly into the transwells (5 µm pore-size) after thawing them and let them recover in there (including medium changes) or would they already start migrating even without a stimulus? Or is there any other way how I could go around the stress of the additional detaching?

Thank you for your help!

Recently I’ve started seeing these clumps in my primary MSC flask (T175). I first noticed these compact clumps in a flask near 80% confluence but I’m also seeing it in less confluent flasks. The clumps vary in size. The medium is neither cloudy nor changing color. I’m using a basic growth media (DMEM, 10% FBS, and 1% antibiotic-antimycotic). When passaging I dissociate the cells using 0.25% trypsi-EDTA for 5 minutes. I’ve been washing the flasks with PBS + 3% antibiotic-antimycotic at media changes, but have not seen any change. Does this look like fungal contamination or could it be cellular contamination (for example, T cells or pluripotent cells clumping) or cellular debris? Whatever it is, it appears to sit a a level above the live, attached cells (with other dead cells).

Thank you!

Hi,

I am culturing primary Motoneuron from mouse spinal cord on Porn and laminin coated coverslips. By day 7 I get about 10% survival of the neurons. The seeding density was 5000 cells. What can I do to increase the survival of the Motoneurons.

please share your thoughts

thank you

I am using propidium iodide (Hi-media Product) but i want to know how much Concentration of propidium iodide is required to stain in 100 cells ? 

I have been trying to do Clonogenic assay for a while now. It has been unsucessful so far standardizing the right number of cells to form colony in a 6 well plate.

Kindly guide me in preparing this

Hi everyone,

I am currently working on BJ human fibroblasts and experienced some troubles in culturing and detaching cells from flasks.

More precisely, I noticed a very slow growing rate ( 100% confluence will probably be reached in 7/10 days in T75 flasks) and I have difficulties in detach them even adding more tryspin and incubating at 37°C up to 10 min. I also tried to agitate the cells by hitting and shaking the flask, but very few cells detached, eventually .

Could you please provide any protocol or tips how to handle this type of cell line?

The splitting passage is now 14.

Thank you all in advance.

Viola

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

Hey Guys I split HeLa cells from a confluent T25 flask into 4 T25 flak. The T-25 flask was over confluent and the media was yellow. I changed the media, gave the cells 1XPBS wash and split them in 1:4 ratio. This was done on 14th of November 2022. Now almost 8 days have passed and the cells haven't reached confluency yet. There are few colonies but I could see most cells are yet to adhere properly.

What should I do?

Thanks and Best

Hi fellow researchers,

I have been using this MSC cell line (hTERT immortalised) for some time. However, they have very different growth pattern (See attached) and I have done test on what factors might affect that (passage number, cell density prior to seeding the cells etc.) but came back empty handed. The pattern seems to appear completely random. Usually the same pattern can be seen if they are plated at the same time. The ideal pattern would be cells growing fully confluent, instead of looking like a web.

I am just wondering if anyone out there might have seen something like that before?

Procedure:

2500 cells were seeded in each wells on a 384-well plate. They were grown for 5 days in AIM-V serum free medium (it's a co-culture system and if I add the target cells and the drugs the MSC would have been growing for 5 days).

Thanks a lot!

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 All,

I am trying to use a technique used by previous graduate students that involves growing yeast in SC media -Uracil buffered to pH 6.75. All the literature published from our lab states that the growth media was buffered to pH 6.75 with 100 mM HEPES.

My problem is that I have tried multiple times to buffer the solution and have not gotten a stable pH through the growth cycle.

Initially, I prepared 1M HEPES buffer pH 6.75 and diluted it 10X to reach 1X media concentration, however upon dilution to 1x, the pH drops significantly. I then prepared the 1X media with the 100 mM HEPES, and adjusted the pH with NaOH and KOH (two separate attempts), however the pH was not stable through during growth and acidified significantly. I then buffered the 1X media with a pH 6.5 and pH 7.5 100 mM HEPES buffer, and the same issues persisted.

Does anyone have any insight into what I an missing here?

Hello, everyone. Mi name is Jorge, and I'm student in Universidad de Guanajuato in Mexico. The last weeks, I have cultured primary leukocytes in DMED with 1% of penicillin-streptomycin, and 20ug/ml of Concanavalin A; but my cells don't increase in number in 24 hours, 48 hours, or 36 hours.

I obtained the cells with Ficoll-hypaque PLUS from healthy patients, and I used P60 dishes.

I hope can you help me! :)

Our cell line keeps detaching from the plate during washing etc, whilst my control HEK293 cells don't. I am considering adding a higher concentration of paraformaldehyde but am unsure how much to add and for how long to incubate it with. We have noted that it is already a very picky cell line as it doesn't grow as fast as HEK293, and it does not survive as a suspension. Any ideas?

Hello! I'm a little bit confused - all of my life I was taught, that cells must be preserved in a mixture of 90% FBS + 10% DMSO (maybe 5% EG + 5% DMSO, for stem cells). But right now I'll have to cryopreserve a lot of cell lines in a big quantity at once. I made a little calculation, and I simply can not spend 0,6 L of serum for this task. For your interest - all of these lines are well characterized and well established cancer cultures.

Some of my colleagues insists that it is completely fine to cryoreserve cancer cells lines (not primary cultures) in a mixture of full growth media + 10% DMSO.

Is that true? Or I'll have a cryostorage full of dead cells at the end?

Dear research community,

I'm beginning to work with the renal rat cell line PC12 and the information I collected so far are confusing.

I read about the doubling time of the cells reaching from 48h to 90h, which is quite a difference when planning experiments.

Also, I got two medium suggestions based on RPMI-1640 and the other on DMEM.

For starters, I just want to cultivate the PC12 cells in suspension and later on differentiate them with NGF.

Looking forward to your answers :)

My intention is to plate the cells in 24 well plates followed by transfection. Although the flasks are 100% confluent, the cell count per square that I'm getting using the hemocytometer is not matching to the exact cell density of the flask before trypsinization.

Usually, I collect cells in 2ml of L-15 medium suspension after trypsin treatment. Then I prepare 20 ul of loading suspension (10 ul of cells and 10 ul of trypan blue dye). To load the cells into the hemocytometer I use 10 ul from the loading suspension. But when I count the cells using a hemocytometer hardly I'm getting less than 30 cells per square. I'm experiencing this issue only in the cell line that I developed. To cross-check this I have used another 100% confluent cell line (EPC) for counting where I'm getting more than 200 cells per square.

Could someone assist me with this? Thanks in advance.

Hi everyone,

I've been recently culturing 4T1 cells. The cells looked happy and healthy after thawing for a few days (i changed the medium every other day); however, after trypsinization, they had abnormal morphology and all died after a day. I've attached two images of the culture just before throwing them away. The culture was also a bit bad-smelling and somehow turbid.

I had the same problem with my other cell line NIH3T3. The cells are in good health initially but develop small particles and brownish clusters after trypsinization, and die after a day or two.

I assume this might be a contamination problem, probably with yeast.

Can anybody tell me what type of contamination they think this is, and how we can get rid of it for our future cultures?

Hi everyone,

I've been recently culturing NIH3T3 cells. After a round of trypsinization and freezing, the thawed cells seem a bit unhealthy. I see random black clusters of cells approximately after 24h of culture, which seem to be dead cell clumps. While I change the medium frequently (like every day), the black clusters are reduced in frequency but a few still seem to be there even after extensive washing (3cc PBS for a T25 flask, x2). They again accumulate after a few hours.

I assume there must have been a problem with trypsin, despite double checking the concentration which was 0.25%. I also kept the centrifugation speed for thawing quite low (about 200 g for 10 minutes); I am pretty confused.

Has anybody had the same problem following trypsinization? And what is your suggestion to revive the living cells in culture and eliminate the dead ones?

The T3 from Sigma T5516-1mg bottle. MW is 672.96 g/mol.

The Spec sheet for the SUM102 PT cells says use 20ug/ml stock concentration to get a final concentration of 6.7ng/ml (10nM) and add 168ul to 500ml medium. T3 has to be diluted in 0.1M NaOH. How to get the required 20ug/ml stock from 1mg total in bottle? The calculations I did required using a vanishingly small mass of T3. I would like to know a better approach to handling complex media supplements. Thank you

Hi guys,

I'm studying the effects of exogenous palmitic acid on cancer cells (cell culture, in vitro).

I performed LDH assay and MTT assay, but these results were that LDH values were higher and MTT values were lower, showing the inhibition of cancer cells.

But some research indicated that PA could promote the proliferation of cancer cells. Actually, I don't know why and maybe there are some different from others?

I'm still looking at papers to find out some solutions but until now I haven't got any ideas. Can anybody tell me something about the role of PA in cancer cells? Many thanks.

To investigate the effects of different drugs on the cancer cell, I did LDH assay and MTT assay. (p.s. The concentration of drugs was constant, I just looked at different drugs or their combination.) Briefly, I seeded cells in a 96-well plate, added drugs, and then put the plate in the incubator. After that, I took out the supernatant into a new 96-well plate to do LDH assay and the 96-well plate which was full of cells was used to do MTT assay. But, these results I got made me confused.

For example, the MTT value of one group was more than 100%, compared with the blank group, indicating cell proliferation. But the LDH %cytotoxicity of this group I mentioned before was about 3%, which meant some cells were killed. Ummm... I was confused about it. cell proliferation and cell death at the same time? The MTT results didn't correspond with the LDH assay.

Can anyone help me explain it? I thought there was something weird and I was wrong. I don't know why the MTT results didn't correspond with the LDH assay.

Btw,

LDH assay kit: CyQUANT™ LDH Cytotoxicity Assay Kit

And LDH %cytotoxicity is calculated according to the protocol.

MTT assay kit: CyQUANT™ MTT Cell Proliferation Assay Kit

MTT cell viability is calculated according to Christian Betzen's answer. https://www.researchgate.net/post/How_do_I_calculate_cell_viability_in_MTT_assay

Many thanks.

I've had 2 cryovials given to me and I can't seem to get the cells to attach & grow. Complete media is 10% FBS, RPMI 1640 w/ L-glut, Sodium Pyruv, HEPES, Pen/Strep, & BME.

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.

Hi guys,

I seeded adherent cells in the 96-well plate (10,000 cells/well) for LDH and MTT test. After overnight incubation, I discarded the medium I added before and used starve medium or DPBS to wash cells and discarded. Then I added starve medium with the chemical compound. After finishing, I observed cells under the microscope and found some cells were washed away.

I used vacuum aspiration to wash cells at first, but I found its power was too strong and many cells were washed. Then I used the pipette to aspirate medium instead of vacuum aspiration and fewer cells were washed than before. However, there were still some cells washed. And actually, I don't know whether the results were influenced by cells that were washed away.

Could anyone else tell me how to wash cells to avoid losing cells? Many thanks.

Btw, I used the 96-well plate from Greiner (Item No.: 655180).

24h? 48h? 72h?

Hi everyone i have come a cross a problem hope that you people will guide me . My question is that i have made a stock solution of 1mg/ml and used concentration of 100ul, 50ul, 25ul and 12.5 μl. Now the concentration i have  used is 100,50,25 μg/μl or 100, 50 and 25μg/ml. 

Thanks in anticipation .

With kind regards

Bilal  Malakzai

Fellow Researchers, I need some wisdom.

I am currently performing an experiment involving CaCo2 cells seeded on a Transwell. I don't have experience with such assays, therefore please forgive me, if my questions are somewhat ignorant. I've searched ResearchGate and found some answers in this discussion:

(23) Can transwell-cultured Caco-2 monolayers be imaged with BD Pathway confocal microscopy while keeping them in transwell? (researchgate.net)

However, I wonder - is there a possibility to observe the Transwells while on a plastic plate, without the necessity of transferring the Transwells to a glass dish? Additionally, I use the type of Transwells that stand on the bottom of the well, not hang on the borders of the well, therefore I am not sure if this solution would work in my case. My idea was- if I used a non-inverted microscope, could I maybe see my cells in the Transwell? I understand that after seeding, my cells need 21 days to differentiate and form a monolayer, however, how can I check that they are in fact attached to the Transwell and growing if I have no way of seeing them? Is staining with some viability dye the only way? I am afraid of a scenario in which I wait 21 days for my cells to differentiate while in fact – they didn’t even attach?

I am seeing white powder (dot like) in cell culture medium. While seeding cells in a flask, they are fine for 2-3 days, and as the time progresses it starts looking a bit stressed. After 5-6 days, i am not able to see any cells, just a powdery layer on cell culture medium, which gets dissolved when we shake the flask. It’s not a fungal or a bacterial contamination. Can anyone suggest what type of contamination it can be or is it something else?

Dear colleagues, I’ve started working with cultured primary neurons and came across a problem.

I need to depolarize neurons for different time intervals (up to 1 hour) and then use them for an assay 2 days after. The problem is that most of them are dying after depolarization.

I culture neurons in complete neurobasal medium (Neurobasal + 2% B27 supplement + 1%Glutamax +1% penstrep) with 1/3 media change every 3 days. I depolarize them on DIV11-14 by swapping media on Tyrode’s solution (45mM KCl) for up to an hour. Then I wash the cells with Tyrode’s solution (5mM KCl) twice and swap the collected media back.

45mM K+ Tyrode’s: 100 mM* NaCl; 45 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 1.04 mM Na2HPO4; 26.2 mM NaHCO3; 10.9 mM HEPES; 10 mM D-glucose

* NaCl is used to adjust osmolarity of the solution, so concentration varies.

Since in the current setup I depolarize cells in 5%CO2 incubator I used buffering formula of Neurobasal media. I adjust the pH to Neurobasal’s pH=7.7 and checked that in CO2 incubator it equilibrates to pH=7.4. And I adjust solution’s osmolarity to match the current neuron’s medium too.

Also, I depolarized neurons in live cell imaging using GCaMP6s to monitor calcium elevation and after minutes I can see that some neurites are destroyed (Fig.1, attached) and after 0.5-1 hour cells don’t look good and most of them die afterwards (Fig.2).

At the same time, I keep seeing papers with no explicit details on solution and osmolarity where cultured primary neurons are stimulated with KCl for hours. For example, here 6 hours of 55mM KCl (https://www.nature.com/articles/nature09033).

I guess there are a lot of people routinely working with primary neuronal culture. Could you please help me, what am I missing?

Hello everyone! I have somehow silly question, but anyway:

I need to perform 2-point calibration for CO2 incubator, but professional tools currently unavailable due to administrative difficulties.

Is it possible to use "candle jar"-technique for 2-point calibration of CO2 level? For zero point I planning to get a room level of CO2 (which is near 0,03%vol), and for second point I'll try to use a point, at which candles would fade.

I heard that candles are stopping to burn at 7%vol of CO2, is that correct?

P.S. I know, that this is a very silly and very imprecise solution, but it is suitable for me.

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.