Rat anti cd45 apc cy7

Cells were stained with Ghost Violet™ 510 Viability Dye (TONBO Biosciences) for dead cell exclusion, blocked with Fc block (2.4G2, BD Biosciences) and stained with the following anti-mouse antibodies from Biolegend: rat anti-CD4-BV785 (GK1.5), rat anti-CD44-PE-Cy7 (IM7), hamster anti-TCRb-APC (H57-597), rat anti-CD8-PerCP-Cy5.5 (53-6.7), rat anti-CD45-APC-Cy7 (30-F11), rat anti-CD25-FITC (3C7), rat anti-CD5-A700 (53-7.3), rat anti-CD127-BV421 (A7R34) and hamster anti-CD69-PE (H1.2F3) (for thymus samples only) or rat anti-CD62L-PE (MEL-14) (for spleen and lymph node samples only). All antibodies were diluted at 1/200, except for anti-CD4-BV785 (1/400) and anti-CD44-PE-Cy7 (1/700). AccuCheck counting beads (Thermo Fisher Scientific) were added to each sample in order to calculate absolute cell numbers. Samples were analysed on a Fortessa (BD Biosciences) instrument.

Suspensions of white blood cells and microglia were transferred to 2 ml Eppendorf microcentrifuge tubes, and a small fraction of sample was removed for single-color controls. To stain dead cells, live/dead violet (1:500–1:1000; ThermoFisher, L34955) was added and incubated for 5 min on ice. Tubes with live/dead-stained cells were topped off to 2 ml with FACS buffer (0.5% BSA HBSS, 1 mm EDTA) and spun down at 300 × g for 5 min at 4°C. Pellets were resuspended and incubated with 1:200 Mouse Fc Block (BD, 2.4G2, 553142) on ice for 15 min. Samples were incubated with 1:200 rat anti CD45-APC/Cy7 (BioLegend, 103115) and rat anti CD11b-PE/Cy5 (BioLegend, 101209) at 4°C. Tubes were topped off to 2 ml with ice-cold FACS buffer and microglia pelleted at 300 × g for 5 min at 4°C. Supernatants were removed and microglia resuspended in 500 μl. Resuspended microglia were filtered through corning strainer polystyrene tubes (Corning, 352235). Flow cytometry data were acquired on Aria II, Fortessa HTS, and LSRII HTS, and analyzed using FlowJo.

For FACS analysis of tumors and control tissues (back skin and abdominal skin, respectively), the tissue was dissected, minced, and digested in a collagenase solution [5 mg/ml Collagenase II (Sigma), 40 µg/ml DNaseI (Roche)] for 30 min at 37°C. The digested tissue was passed through a cell strainer before erythrocyte lysis with PharmLyse (BD). After washing and a second filtration step, the cell suspension was labeled with fluorescently tagged antibodies [hamster anti-podoplanin-PE (1:400, clone 8.1.1, eBioscience), rat anti-CD31-APC (1:300, clone MEC13.3, BD), rat anti-CD45-APC/Cy7 (1:200, clone 30-F11, Biolegend), and rat anti-PDL1-PE/Cy7 (1:200, clone 10F.9G2, Biolegend)]. 7AAD (Biolegend) was used for life/dead discrimination. Inguinal LNs were processed essentially as described before (31 (link)). In brief, the capsule of dissected LNs was ruptured and the tissue was digested with 1 mg/ml Collagenase IV (Gibco)/40 µg/ml DNaseI for 20 min at 37°C to release the majority of the immune cells. The remaining stromal fragments were washed twice, digested with 3.5 mg/ml Collagenase IV/40 µg/ml DNaseI for 15 min at 37°C, and disaggregated by pipetting in presence of 0.5 mM EDTA. After filtration, the stromal cell enriched cell suspension was stained as described above. Data were acquired on a FACS CANTO (BD) and analyzed using FlowJo (Treestar Inc.).