Anti cd45 bv605

Dissociated lung cells were resuspended in 100 μL staining buffer and blocked with anti-CD16/32 antibody (BioLegend) followed by staining using fluorescently-tagged antibodies: anti-CD45 BV605 (BioLegend), anti-CD3 PE-Cy7 (BioLegend), anti-CD11b (BioLegend), anti-IA/IE BV650 (BioLegend), anti-CD11c (BioLegend), anti-CD24 AF488 (BioLegend), anti-CD193 PE (BioLegend), anti-GR1 AF700 (BioLegend), anti-CD64 APC (BioLegend), anti-Siglec-F PE-CF594 (BD Bioscience), along with a viability indicator Ghost Dye Violet 510 (Tonbo Bioscience). The list of antibodies, sources, and identifier (Cat#) information is summarized in the key resources table. The data acquisition was performed in LSRII Fortessa by collecting a total of 100,000 events (∼90% single cell events/samples) and analyzed using FCS Express 7 software. Dead cells were excluded based on Ghost dye⁺ (dead cells) and Ghost dye¯ live cells were included for further gating. Compensation for each flow cytometer experiment was performed with unstained and all single-color controls using OneComp eBeads (Thermo Fisher Scientific). Representative dot plots of a multicolor flow cytometry panel used to identify myeloid cell subsets in lung tissues from mice were provided as described (Figure S2).⁶⁰ (link)

Human salivary gland tissues were processed into single-cell suspensions as described above and then filtered through a 35 μm nylon mesh (Corning). Cells were resuspended in phosphate-buffered saline supplemented with 0.1% bovine serum albumin, 10 mM HEPES, 25 mM EDTA, and 10 μM Y-27632 to avoid anoikis of epithelial cells. Single-cell suspensions were preincubated with human TruStain FcX (#4322302, Biolegend), and stained with primary antibodies for 30 min at 4 °C. Dead cells were stained with zombie violet (#423114, Biolegend) and gated out. Stained cells were analyzed using LSRFortessa or sorted using FACSAria II (BD, Franklin Lakes, NJ, USA). Data were collected via FACSDiva software (BD) and analyzed using the FlowJo software. The following antibodies were used; anti-CD45-BV605 (#368524, 1:50, Biolegend), anti-CD31-FITC (#303104, 1:20, Biolegend), anti-CD49f-PE-Cy7 (#313621, 1:20, Biolegend), and anti-CD26-PE (#302705, 1:20, Biolegend).

A suspension of stimulated splenocytes (2 × 10⁵ cells/well) was applied to flat-bottom 96-well plates (Nunc) with previously prepared single-layer MC38 cell culture (the ratio of tumor cells to splenocytes was 1 : 5). APC conjugated anti-CD107a monoclonal antibody, PMA (50 mg/ml, Sigma-Aldrich), ionomycin (1 μg/ml, Sigma-Aldrich), and rh IL-2 (200 U/ml, ImmunoTools) were added to the wells. After 2 h at 37°C, cells were harvested and labeled with fluorochrome-conjugated monoclonal antibodies: anti-CD45 BV605, anti-CD4 FITC, anti-CD8 APC-Fire, and anti-NK1.1 PE-Dazzle (all from BioLegend). Cells were incubated with antibodies for 45 min at 4°C. To identify dead cells, 50 μl of DAPI dye solution (1 μg/ml, Molecular Probes) was added to the samples immediately before analysis. Samples were analyzed by a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software 8.0 and the NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.).