Apc conjugated anti c kit

Fluorescence-activated cell sorting (FACS) purification was performed on cultured cells to remove contaminating feeder cells prior to RNA extraction or subsequent analyses or western blot. Following culture and collection as described above, samples were washed in 2% v/v FBS in PBS and stained with phycoerythrin (PE)-conjugated EpCAM (1:300, eBioscience, clone G8.8) for 30 min at 4 °C prior to washing and resuspension in 2% v/v FBS in PBS. DAPI was added to final cell suspensions for live/dead cell discrimination. Spermatogonia were purified according to their expression of EpCAM^{10 (link)}. For cultured cells expressing constructs tagged with GFP or tdTomato, spermatogonia were isolated based on endogenous fluorescence of these tags, without staining for EpCAM. Isolation of spermatogonia from testes of control and Plzf^–/– mice was performed following staining with allophycocyanin (APC)-conjugated anti-cKIT (Biolegend, clone 2B8, #105812), PE-conjugated anti-CD49f (Biolegend, clone GoH3, #313612), PE-Cyanin7-conjugated anti-EpCAM (Biolegend, clone G8.8, #118216) and fluorescein isothiocyanate (FITC)-conjugated anti-CD9 (eBioscience, clone KMC8, #11-0091-82)^{12 (link)}. FACS was performed at Monash University FlowCore using a BD Influx Cell Sorter (BD Biosciences).