Ears were dissected into small pieces and digested in 500 μl RPMI containing 10% FCS and 1 mg/ml Collagenase P (Roche Diagnostic) for an initial 1 hr at 37 °C. The digested tissues were briefly spin at 2,000 rpm for 5 min and the 200 μl clear supernatant was collected and stored at -20 °C for LEGENDplex analysis. The remaining tissue pellets were resuspended thoroughly with another 1 ml of RPMI + Collagenase P with another 1 hr incubation at 37 °C, and 100 µg/ml DNase I (Roche Diagnostic) was added for the final 30 min. The digested samples were passed through 70-μm cell strainers (BD Biosciences) and the digestion was stopped with 500 µl cold 10 mM EDTA in PBS, and a single cell suspension was obtained in FACS cell staining buffer (BioLegend). Auricular lymph nodes were harvested and meshed through 70-μm cell strainers (BD Biosciences) to obtain single cell suspensions in FACS cell staining buffer (BioLegend).
Chen Y.L., Ng J.S., Babu R.O., Woo J., Nahler J., Hardman C.S., Kurupati P., Nussbaum L., Gao F., Dong T., Ladell K., Price D.A., Duncan D.A., Johnson D., Gileadi U., Koohy H, & Ogg G.S. (2023). Group A streptococcus induces CD1a-autoreactive T cells and promotes psoriatic inflammation. Science immunology, 8(84), eadd9232.
+ Open protocol