Why cells are dying in the centre of some random wells in a 96-well plate?

Hi,

I'm having some troubles when seeding cells in m24 plates: after 24-48 hours of seeding, cells don't seem to attach in some areas of the wells or they attach at the beginning but then they die. This leads to irregular replicates and no-reliable results. I've observed this in 2 different cell lines (sw620 and DLD1), I've changed medium bottles, tried with different cell vials, and put plates in 2 different incubators, but I still have this irregular attachment/growing. Could you help me? I've done lots of this experiments before, both in m24 and m96, and everything went OK. I don't know what can be failing now...

Thank you!

Hi everyone,

I'm having cell growth problem in outer wells of 96 well plates for bioactivity assay, recently.

The problem occurs during the incubation in the 37oC CO2 incubator. While 96 well plate is treated with the cells, firstly 200 ul PBS is added into the outer wells of 96-well plate as shown in the attached figure (wells colored in grey). Afterwards, cells is seeded into each well of the 96-well plate so that each sample can be treated in triplicates, as shown in the attached figure (wells colored in white) then plate is treated with the standard/test solution. After the treatment plate is incubated in the 37oC CO2 incubator for 72 hrs. By the way, I have used these seeding protocol other times, so I know they work.

During this incubation, no growth is observed in the outer wells containing cells (e.g. B2, B3,C2, D2, E2, F2, G2.). To overcome this problem, PBS is added to the gap between two wells too. Still, the problem was not completely solved. I think the problem is caused by evaporation in the incubator. If you may have an opinion about the reason of this?

I look forward to hearing from you.

Thanks in advance :)

I have treated sample OD Value, Solvent control OD value and controlled OD value  

Hi. We have been observing this strange cell death morphology during months. Cells die and acquire a "fried egg" morphology as it is shown in picture (A431 cell line). When it happens, all the cells die alike. It appears randomly in cell plates and not in all the experiments, but we are thinking that affected-wells distribution could be repeated, but it does not occur in all the plates. It has nothing to do with cell treatment, because it has been observed also in control cells. If you have some advice, please anwer :)

Thank you!

I am new to cell culture. I have worked with MIN6 and INS-832 cells. While changing media in a 96-well plate, I tend to end up with a ring shaped gap in the wells. It seems this happens when I remove the media. I tilt my plate, and remove media from the right side using a glass pipet with suction. When media is removed, the middle of the well runs out of media first and I noticed that this "media-less" circle is the same shape I see in the microscope where the cells have left a gap. I can't be the only one who has come across this problem right? Are my cells drying out in that area when I remove media? Is there a pipetting technique or a way to ensure that when I remove the media, this does not occur?

I have passaged my primary fibroblast cells into 6 well plates, in mem 20% fbs without antibiotic, they grew fine, 2 days later I changed the media and immediately some cell started dying and looking different ( picture shows bubble like structures and broken cell wall) , this happens only in certain parts of the same well while in the other areas cell are looking fine (check the other picture from a different part of the well). any help regarding this matter is appreciated

I work on MC3T3-E1 cell lines. The cells appear absolutely fine in flasks, but when I seed them in 12/24 well plates for mineralization (nunc thermofischer plates), then cells appear to die in few of the wells while being absolutely fine in others.

The morphology looks like as if after attachment, media was removed from the wells for cells to become dehydrate and die.

Please find the pictures attached.

Could you please try to help me to get possible explanation for this morphology?

I like to know whether it is possible just to wash out trypsin with PBS, centrifuge followed by resuspending the pellet with serum free medium for cells to be cultured with serum free medium? 

Hi! Over the past weeks, I have observed an anomaly in one of the cell lines I routinely culture (CHSE-214 cells, derived from Chinook salmon). My routine culture cells are grown in flasks and appear to be perfectly healthy. Lately however, I have noticed that when using these cells to seed 24-well cell culture plates, the majority of the cells will die within just a week or two. After a couple of days, the cells start rounding up, detach from the monolayer (they are adhesive cells) and float to the surface of the medium, often forming large clumps of dead cells.

Since then, I have collected cell lysates and performed mycoplasma tests and also QPCR tests for every disease that we routinely work with in our lab. Every one of these tests was negative. What is strange is that I do not observe this in any of my culture flasks. It only seems to happen as soon as I plate them, and they only recently started to do this. I have read that this can happen when too many cells are seeded in the wells and are too confluent, but I only seed my cells at about 70% confluency and count them every time before plating them, so I do not think that is the issue.

Last week I seeded some cells in 3 different types of cell culture plates (tried 2 different brands of TC treated plates and also Corning cell-bind plates). I have not observed any difference between these 3 brands of plates; the cells keep dying. I have also screened the growth media I am using for mycoplasma and some viruses, all of which were negative. There is nothing visible under the microscope either, so I am not suspecting bacterial contamination, especially since the cells appear healthy in their culture flasks and I use the same media bottle for all my cultures. 

We work with a few other cell lines in the lab as well, and all of them perform well when seeded to culture plates. It is just this one cell line, and I was not observing this until a few weeks ago. Anybody have any ideas? I am stumped!