Perm wash in pbs

Cells were fixed in 1% ice-cold paraformaldehyde (10% stock, EM-grade; Electron Microscopy Sciences) and permeabilized in 1 mL ice-cold 80% methanol, as described previously (Zwollo et al., 2010 (link)). After overnight incubation at −20° C, cells were either resuspended in permeabilizing solution (BD perm wash in PBS, BD Biosciences) and stained as described previously, or refixed and stored in FBS containing 10% DMSO at −80C (Zwollo et al., 2010 (link), MacMurray 2013 (link)). Approximately 30,000 events were acquired per sample using a BD FACSArray (BD Biosciences). Duplicate samples for each staining combo were run for each experiment. Contour graphs were generated using WinMDI 2–8 (J. Trotter 1993–1998) software. Contour graphs are shown as log algorithms with intervals of 50%.

Cells were fixed in 1% ice-cold paraformaldehyde (10% stock, EM-grade; Electron Microscopy Sciences) and permeabilized in 1 mL ice-cold 80% methanol, as described previously (Zwollo et al., 2010 (link)). After overnight incubation at −20° C, cells were either resuspended in permeabilizing solution (BD perm wash in PBS, BD Biosciences) and stained as described previously, or refixed and stored in FBS containing 10% DMSO at −80C (Zwollo et al., 2010 (link)). Approximately 30,000 events were acquired per sample using a BD FACSArray (BD Biosciences). Duplicate samples were run for each experiment. Contour graphs were generated using WinMDI 2–8 (J. Trotter 1993–1998) software. Contour graphs are shown as log algorithms with intervals of 50%.