I am expressing GPCR in CHO cells, in suspension and adherent format. But when single sorting the cells using FACS Aria, i have obtained clones for the suspension format, but the adherent format either does not yield any single cells in the 96 well plate or very few clones are generated which loose adherence/ expression upon expansion. The sample preparation protocol is similar for both formats, except that the adherent cells are detached using Trypsin, before the staining is done.
some GPCR may be affected by trypsin. I used to detach the cells with a non-enzymatic cell dissociation buffer, before performing functional assays.
Would any other technique like limited dilution work better for adherent cells rather than FACS sorting?? My colleague has tried limited dilution for suspension cells and it worked fine. But he got fewer number of monoclonal colonies as compared to the number of single cell colonies you can get from a FACS sorting.
Trypsin may be the reason for decrease in cell surface expression. But it would be a good idea to check expression of GPCR before and after trypsinisation and confirm.
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