I have transduced mammalian cells with lentivirus to express an exogenous gene. The transduced cells were sorted out on a FACS machine based on the immunostaining of the protein encoded by the virus, which is expressed on the cell surface (supposed to be ~100% positive). However, when I checked the expression by immunostaining again couple days after sorting, the positive cells were less than 90%, and this percentage kept decreasing in the following days. What could be the reason for this loss of positive cells? Anyone has similar experience or suggestions on this? Thanks.
Adam,
how do unsorted cells behave? Can you detect your protein there after a few days?
Hi Adam,
Speaking transkationally, when you set up your sort, it is best to set a high stringency gating strategy where you sort for only the highest expresses, not the low and intermediate. You should also ask the sort technician to discriminate against dead cells based on fsc/scc as well as foublets. This should reduce the problem
Wolfgang Schechinger The unsorted cells also gradually lost the transgene expression.
>>The unsorted cells also gradually lost the transgene expression.
So it might not be a sorting problem. When your cells divide, they might dilute the plasmid or not infected cells are dividing faster.
Wolfgang Schechinger I agree with you that it is not due to the sorting. Sorting here is just to make sure I have 100% positive cells, so that the population would not be dominated by the non-infected cells which might grow faster.
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