Bv421 anti human cd45

Single frozen vials of the SVF were thawed at 37 °C in a water bath and the content was transferred to 9 mL of RPMI 1640 GlutaMAX medium supplemented with 10% FBS and 1% AA (all from Gibco) before centrifugation at 300 g for 5 min. Cells were incubated first with human Fc receptor blocking solution (1:10, Biolegend, 422302) and efluor 780 fixable viability dye (1:1000, eBioscience, San Diego, CA, USA, 65-0865-14,) in PBS for 5 min at 4 °C. Afterwards, the antibodies were directly added on the cell suspension and incubated for 15 min at 4 °C. For staining of LECs, the following antibodies were used: BV421 anti-human CD45 (1:200, Biolegend, 304032), FITC anti-human CD31 (1:20, BD Pharmingen, NJ, USA, 555445,), Pe-Cyanine 7 anti-human podoplanin (1:200, Biolegend, 337014) and PE anti-human Nectin-2 (1:200, Biolegend, 337409) or the isotype PE anti-mouse IgG1 (1:200, Biolegend, 400114). For staining of DCs, the following antibodies were used: BV421 anti-human CD45 (1:200, Biolegend, 304032), APC anti-human CD86 (1:200, Biolegend, 305411), FITC anti-human HLA-DR (1:200, Biolegend, 307603) and PE anti-human Nectin-2 (1:200, Biolegend, 337409) or the isotype PE anti-mouse IgG1 (1:200, Biolegend, 400114). Cell suspensions were washed once prior to recording on a CytoFlex S instrument (Beckman Coulter). Data were analyzed using FlowJo software version 10.8.1 (BD Life Sciences).