BM, spleen, and cells recovered from the ear dermis from infected and naïve mice were analyzed by flow cytometry. BM was harvested by flushing tibiae and femora in phosphate-buffered saline (PBS) solution and the cells were then passed through a 25-gauge needle to obtain a single cell suspension. Spleens were manually dissociated in PBS. Ear tissue homogenates, prepared as described above, were filtered in 5 mL round bottom polystyrene FACS tubes with 35 μm nylon mesh-screen filter before flow cytometry staining. PBS was supplemented with 0.1% bovine serum albumin (BSA) and 0.5 mm ethylene-diaminetetraacetic acid (EDTA) for flow cytometry staining, and non-specific staining was blocked using unlabeled anti-CD16/CD32 antibody (Fc block). Antibody combinations were modified from previously published studies, including by our group [16 (link),27 (link),28 (link),29 (link)]. Please see Table S1 for a complete list of antibodies. Cells were fixed with 2%PFA in PBS prior to acquisition of samples using a 4-laser LSR Fortessa (BD Biosciences, Mountain View, CA, USA). Data were analyzed using FACS DiVa software (version 8.1).
Free full text: Click here
