▲ CRITICAL STEP Not all sorted cells may survive the FACS process or the subsequent outgrowth; therefore, the number of plates prepared and cells sorted may need to be adjusted to ensure an adequate number of clonal lines derived. 56Preparation of cells for FACS. Dissociate the cells (from Steps 11 or 28) by aspirating the medium completely and adding enough TrypLE to thinly cover the adherent layer of transfected cells. Incubate the mixture for 5 min at 37 °C and add 400 μl of warm D10 medium.57Transfer the resuspended cells into a 15-ml Falcon tube and gently triturate 20 times.
