I have cultured PBMC and some RBC contaminant for 2 weeks in suspension and low attachment plate. From the very first day, I noticed that there are many cells that have already turned all black-ish or grey-ish color. I don't know why and I keep continuing my culture process because the sample is very limited and I don't want to waste any samples. This is very confusing as because I will do viability assay using trypan blue, but the cells were already black. So I can't see the blue-ish color that supposed to appear. For additional information, my medium is DMEM F12, EGF, FGF, CoCl2, ITS, Penstrep, and Amphotericin.
PBMC don't turn black. You have a very strange problem. Either your microscope is damaged, or a very weird molecule got into your culture somehow. How much cobalt is that?
PBMC do largely die over time, so it's difficult to estimate what portion might be alive based on other people's experiments. Can you access a Hoffman's contrast microscope? With that, you could discriminate live from dead PBMCs without any staining, by just looking at them. The live ones look nice and spherical, and the dead ones flat and kind of crinkled.
You could also use a dye like MTS to tell if there are any live cells at all in your strange culture.
John Schloendorn thank you for your response. I believe that there is nothing wrong with my microscope. I use 100 uM CoCl2 because I want to stabilize the HIF-1a to induce hypoxia response in the cell. Also, I don't have access to that microscope.
Hello Rafli, Have your cells lost size and become pyknotic/speckled? Do you need to supplement with Amphotericin?
I have found that cell growth is significantly impact by the presence of antifungal reagent. Have you titrated the [cobalt], I would assume that the hypoxic simulation via the ion would be deterimental to cell viability. I would start with a titration of the cobalt in culture medium without amphotercin supplementation.
best of luck
Martin Waterfall thank you for the response. I use amphotericin because that is what I know so far based on the literature. However, there is no standard medium for the culture. Moreover, the cells are limited so I was worried about any possible contaminations. Also, i believe that my cells size stays the same. I dont know about the pyknotic/speckled nucleus because I haven't tried to do immunofluorescence.
Rafli, should be able to see the speckling through the cytoplasm under conventional microscopy. I have found exposure to cobalt can be quite toxic to other cellular material.
Martin Waterfall this is what my cells looks like under 400x magnification. Does this may suggest that the cells become speckled?
Hello Rafli, your micrograph does appear to illustrate apoptosis in your cultures. I can's see any serum in your DMEM usulaly ~10%FBS? I would also suggest that you remove the amphotericin and titrate the cobalt, as I would anticipate both reagent are stressing/killing the cultures.
best wishes
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