MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2

McCarthy, Mary K.; Reynoso, Glennys V.; Winkler, Emma S.; Mack, Matthias; Diamond, Michael S.; Hickman, Heather D.; Morrison, Thomas E.

Single cell suspensions were incubated with anti-mouse FcγRIII/II (2.4G2; BD Pharmingen) for 20 min on ice and then stained with antibodies against the following cell surface markers in 1X PBS/2% FBS for 1 h on ice: CD45 (30-F11), CD11b (M1/70), Ly-6C (HK1.4), Ly-6G (1A8), CD19 (6D5), B220 (RA3-6B2), TCRβ (H57-597), CD4 (RM4-5), CD8 (53-6.7), CD95 (Fas; 15A7), GL7 (GL7), IgD (11-26c.2a), and CD138 (281-2) (all Abs from BioLegend, BD Bioscience, or Thermo Fisher). Cells were washed three times in PBS/2% FBS and fixed overnight in 1X PBS/1% PFA prior to analysis. For intracellular analysis of iNOS expression, cells were stained first with antibodies against cell surface markers, fixed for 15 min at RT in PBS/1% paraformaldehyde (PFA)/0.1% saponin, and then stained with anti-iNOS Ab (clone CXNFT; Thermo Fisher) in PBS/2% FBS/0.1% saponin for 1 h at 4˚C. Cells were washed three times in PBS/2% FBS/0.1% saponin, fixed overnight in 1X PBS/1% PFA, and analyzed on a BD LSR Fortessa cytometer using FACSDiva software. Further analysis was performed using FlowJo software (Tree Star).