Anti mouse gl7

The draining inguinal LNs were homogenized and filtered through a 70-μm cell strainer, and 2×10⁶ cells were stained with the viability dye Near-IR and blocked with anti-mouse CD16/32 (S17011E clone, BioLegend, USA). Subsequently, the cells were stained with anti-mouse CD19 (Brilliant Violet 605™, 6D5 clone), anti-mouse GL7 (PE, GL7 clone), anti-mouse Fas (APC, SA367H8 clone), anti-mouse CD4 (FITC, RM4-5 clone), anti-mouse PD-1 (Brilliant Violet 421™, 29F.1A12 clone), anti-mouse CXCR5 (PE/Cyanine7, L138D7 clone) and anti-mouse CD3 (PerCP/Cyanine5.5, 17A2 clone), which were all purchased from BioLegend. After staining, the cells were fixed, resuspended in PBS, and then acquired on a BD FACS Canto™ flow cytometer.

Cell suspensions were incubated for 20 min at 4 °C in PBS containing 1% FCS and 10 mM EDTA with the following antibodies: anti-mouse CD45, anti-mouse CD31, anti-mouse PDPN, anti-mouse CD21/CD35, anti-mouse SCA1, anti-mouse B220, anti-mouse CD4, anti-mouse CD8, anti-mouse CD19, anti-mouse CD38, anti-mouse GL7 and anti-mouse CD11b (all from BioLegend); anti-mouse CD157 and anti-human CD45 (both from BD Biosciences); anti-human PDPN and anti-human CD31 (both from Thermo Fisher Scientific); and anti-human EPCAM, anti-human CD14, anti-human CD3 and anti-human CD19 (all from BioLegend). LIVE/DEAD cell discrimination was performed either by using a fixable BV510 Dead Cell Staining Kit (Molecular Probes) before antibody staining or by adding 7-aminoactinomycin D (Calbiochem) before acquisition. Cells were acquired with an LSR Fortessa (BD Biosciences) and analyzed with the FlowJo (v.10) software (FlowJo LLC) according to established guidelines. Cell sorting was performed using a BD FACSMelody Cell Sorter and the FACSChorus (v.1.3) software (BD Biosciences).