Gatelogic software

Splenocytes isolated from each mouse were stained with antibodies to surface or intracellular antigens and incubated for 30 min at room temperature. Labeled cells were washed and suspended in 150 µL of PBS and evaluated by flow cytometry, as previously described [14 (link)]. In brief, intracellular antigens were stained by incubating 500 µL suspensions of the cell pellets with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) in the dark for 45 min. Cells were then treated with permeabilization buffer (eBioscience, San Diego, CA, USA), and incubated with an intracellular antibody mix for an additional 30 min at 4 °C. All antibodies were obtained from eBiosciences and Biolegend (San Diego, CA, USA) (Supplemental Table S1). Supplemental Table S2 lists the immune cell types analyzed as well as corresponding fluorochrome-labeled antibodies used for analysis. Flow cytometry was performed with a CyAn ADP Analyzer (Dako, Ft Collins, CO, USA). Gatelogic software (eBioscience) was used for data analysis.

Mouse splenocytes were isolated as previously described [12 (link)] with a final concentration of 5×10⁶ cells/mL. S1 Table lists the immune cell types analyzed and corresponding fluorochrome-labeled antibodies. Cells were mixed with permeabilization buffer (eBioscience, San Diego, CA, USA), centrifuged, resuspended and analyzed by flow cytometry as previously described [12 (link)] using a CyAn ADP Analyzer (Dako, Ft Collins, CO, USA). Flow data was analyzed using Gatelogic software (eBioscience).