Single Cell Phospho-Flow Cytometry

Single cell signaling analysis was carried out using phospho flow with fluorescent cell barcoding as described¹⁹ . Briefly, B cells were treated as described in the figure legends before fixation (BD Phosflow Fix Buffer I, BD Biosciences, Franklin Lakes, NJ, USA). The cells were then washed three times with PBS and stained with different concentrations of the barcoding reagents Alexa Fluor 488 5-TFP, Pacific Blue Succinimidyl Ester and Pacific Orange Succinimidyl Ester (all from ThermoFisher Scientific, Waltham, MA, USA) for 20 min at room temperature. The cells were then washed twice with flow wash (PBS, 10% FCS and 0.09% sodium azide), combined in one tube and permeabilized with BD Phosflow Perm Buffer III (BD Biosciences) which was pre-cooled at −20 °C, and kept at −80 °C until further processing. Before antibody staining, the cells were washed three times with flow wash. The antibodies used in this study are listed in Supplementary Table 1, including the surface marker anti-CD19. After 30 min incubation with the antibodies, the cells were washed once, resuspended in flow wash and analyzed with a BD FACSCanto II (4-2-2) cytometer equipped with 405 nm, 488 nm and 633 nm lasers. The data were analyzed in Cytobank (https://cellmass.cytobank.org/cytobank/) as described^{4 (link)}.