Fluorofix fixation buffer

Specific direct labeled antibody to cell surface antigen CD20 (BioLegend, San Diego, CA) was used to assess splenocyte population. Following primary fixation in Fluorofix fixation buffer (cat 422101, BioLegend) cells were resuspended in permeabilization buffer (cat 421002, BioLegend). Survivin antibody (clone 60.11 FITC, Novus Biologicals, Littleton, CO) was added. Cells were washed in permeabilization buffer (cat 421002, BioLegend) followed with a final PBS wash and resuspension in Fluorofix fixation buffer (cat 422101, BioLegend). Sample acquisition was obtained on a Core facility BD FACSCalibur flow cytometer running CellQuest software followed with analysis using FCS express software. Analysis was based upon isolated gating of lymphocyte populations and co-localization of survivin with specific CD markers as indicated.

Skin infiltration of autoreactive PMEL CD8⁺ T cells in the footpad was quantified by flow cytometry (Cytex Aurora cytometer). Footpad skin was harvested at week 4 after vitiligo induction and treated with RPMI-1640 media (Gibco, #11875093) containing 2.5 mg/mL of collagenase IV (Sigma-Aldrich, #C6885) and 1 mg/mL deoxyribonuclease I (Sigma-Aldrich, #DN25) at 37 °C for 45 min. Skin samples were manually crushed through a 100-μm cell strainer (Fisher Scientific, #22363549). The following dye and flow cytometry antibodies were used for staining CD8⁺ and Thy1.1⁺ double-positive PMEL T cells that originated from the donor mice: TruStain FcX PLUS (anti-mouse CD16/32, Biolegend, #156604), Zombie Aqua™ Fixable Viability Kit (Biolegend, #423101), APC-Cyanine7 anti-mouse CD45 (Biolegend, #103116), PerCP-Cyanine5.5 anti-mouse CD8b (Biolegend, #126610), and FITC anti-mouse CD90.1 (Thy1.1, Biolegend, #202503). Cells were fixed by FluoroFix™ fixation buffer (Biolegend, #422101) prior to flow cytometry analysis.