▲ CRITICAL Isolation of clonal cell populations from the transfected cells (Step 51) can be performed 24 h after transfection by FACS (Steps 54–65) or by serial dilutions (Steps 66–70). Given that cell types can vary greatly in their response to FACS, clonal-density dilution or other isolation procedures, literature specific to the cell type of interest should be consulted. 54Preparation of FACS media. Cells can be sorted in regular D10 medium supplemented with penicillin-streptomycin. The antibiotics-containing medium should be filtered through a 0.22-μM Steriflip filter. If fluorescence sorting is also required, phenol red–free DMEM or DPBS is substituted for normal DMEM.55To 96-well plates, add 100 μl of D10 medium supplemented with penicillin-streptomycin per well.
