My project is based on isolating the cancer stem cells and studying them. After using CD44 and staining them in KGM media, FACS sorting was done. The sorted cells were found to die within 2 days. We have tried it twice. Why is it happening?
Which solution did you use in FACS sorting process? Did you check the morphology of sorted cells after isolation?
It is very important to check that there are no preservatives in the antibody labeling. e.g. sodium azide.
secondly you have to work very quickly to prevent oxidation of the medium in which the cells are sorted.
Finally many antibodies can induce apoptosis, to check on the cell itself before sorting.
I agree with Holger, many cells do not like being alone and will not grow very well as single cell clones. Rock inhibitors will increase cell proliferation, but the downside is that the cells de-differentiate and so your phenotype is different. Try culturing with a 50:50 mix of media and 3T3 conditioned media.
Good Luck
Hi Holger
can you tell me something about the concentration of Y-27632 . dihydrochloride as a culture media supplement
@holger fey : thank you for replying sir. We dint use ROCK inhibitor.
@dung truong : The cells were fine for 24 hours but then they started dying and we tried replacing the media. still dint work.
I'm not familiar with the cell line or which flow cytometer you are using. You can narrow down the source of the problem by taking aliquots of cells at different steps. For example: a batch that has been harvested but not stained with antibody, stained batch that was not sorted, a stained batch not sorted but left at the same conditions as the sorted cells (like on ice for the same amount of time). Plate the cells in the same media you are plating your sorted cells, ideally at a similar cell density. You could even plate a few different densities to see if that helps. As annoying as it can be to run an experiment just to find the source of a problem, this will let you know which part of the processing you need to focus on.
i understand its an annoying process :) but yes i have to find the solution ... thanks fr the help allison
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