Isolation of Splenic Mononuclear Cells

Splenic mononuclear populations were isolated as previously described (Montes de Oca et al., 2016a (link); Stanley et al., 2008 (link)). Briefly, mice were sacrificed by CO₂ asphyxiation after which the spleen was excised, weighed and collected into 10 mL 2%FBS.PBS (2% (v/v) FBS in 1x PBS). Spleens were passed through a 100 μm cell strainer (Falcon) using the back of a 5 cc/ml syringe plunger (Terumo Medical). Cells were resuspended in media and centrifuged at 350 g in an Eppendorf Centrifuge 5810 R (Fisher Scientific, Thermo Fisher Scientific). Cell pellet was incubated in 1 mL Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich) for 6 minutes at RT. Cells were washed again in 10 mL 2%FBS.PBS after which cell pellet was diluted in DPBS (GIBCO) and Trypan Blue Stain (Invitrogen) and counted using Countess Cell Counting Chamber Slides on the Countess II FL (both from Invitrogen), as per manufacturer’s protocol.