Single-Cell Isolation and Staining Protocol

Mouse tissue were harvested and immediately homogenized with the McIlwain Tissue Chopper (Ted Pella, Inc.) to obtain a single-cell suspension as described previously [41 (link)]. Briefly, mechanically homogenized tissue was digested in RPMI 1640 medium containing 0.525 mg/mL collagenase Type 1 (Worthington Biochemical Corporation), 0.02 mg/mL Deoxyribonuclease I (Sigma), and 10% FBS in a 37°C shaker for 1 hour at 250 rpm. The homogenates were passed through a 70 μm filter and red blood cells were lysed with RBC Lysis Buffer (0.034 M aluminum chloride, 0.01 M potassium bicarbonate, 0.1 mM EDTA in dH₂O) for 30 sec. Cells were suspended in flow wash buffer (1% BSA and 0.1% sodium azide in DPBS) and an equal number of cells subjected to flow cytometry. Cells were first incubated with Chrom Pure Mouse IgG (Jackson Immuno Research) to block Fcγ receptors. Cells were then stained with extracellular antigen antibody on ice for 30 minutes prior to permeabilization/fixation on ice for 20 minutes and intracellular staining on ice for an additional 30 minutes. Cells were washed and measured with the BD LSR II flow cytometer (BD Biosciences). Data was analyzed using FCS3 Express software (De Novo).