I have 3T3 cells seeded onto 24-well plates with a density of 6000 cells/well in MDEM containing 10% BCS and 1/200 streptomycin-pencillin. The cells were incubated for 72 hours at 37 degrees in 5% CO2 until confluence reached about 60%.
I was about to perform an assay that required the addition of bacteria to the wells, so I changed the media to an abx-free MDEM, containing the same 10% BCS but no antibiotics. I noticed that almost instantly, many cells started to shrink and atrophy ("look unhealthy"), and the cytoplasm appears to become dotty and full of tiny granules. Interestingly, this atropy was mainly limited to cells on the right side of the 24-well plate. The cells do not stain well with Giemsa stain, compared to healthy 3T3 cells. Any suggestions as to what might be the issue? I've attached two images highlighting the differences.
My supervisor did the same experiment using the cells I prepared, and did not experience any issues using the same abx-free MDEM.
Plate drying is a strong possibility. The instantaneous change in morphology is the least likely due to Abx withdrawal. The guidance suggested by Ms.Dacie appears to be the most practical.
I agree. The cells rather should be more comfortable without antibiotics.
I've been troubleshooting for a while to no avail. The problem is not with the media, and in fact, I can change the media reliably without damaging the cells. However, when I try to wash the cells with PBS and then fix them with 10% formalin in PBS, I get the same shrinkage of cells.
Drying is not likely to be problem as the liquid in the wells was replaced within the span of 2-3 minutes. The reagents are not contaminated as my supervisor used them without issues. I have tried to be gentle by adding the PBS by dripping it down the side of the well instead of dropping them directly on top of the cells. Any ideas? I'm suspecting it's something very minor that I overlooked completely.
I've attached an image that better illustrates how my cells look like.
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