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.

The left popliteal LN was gently homogenized in a Biomasher II tissue homogenizer (Kimble-Chase) in RPMI 1640 (HyClone) supplemented with 10% FBS. For flow cytometric analysis of infiltrating footpad or dLN cells, 2.5 mg/ml collagenase type I (Worthington Biochemical) and 17 μg/ml DNase I (Roche) were added to RPMI 1640 with 10% FBS, and samples were incubated for 1 h at 37˚C with shaking (130 rpm). Following erythrocyte lysis (footpad only), cells were passed through a 100 μm cell strainer (BD Falcon) and total viable cells were determined by trypan blue exclusion. For analysis of circulating immune cells, blood was collected from the inferior vena cava into 50 μL heparin. Following erythrocyte lysis, cells were stained as below.