Cd8a pe cy7

Flow cytometric analysis was performed as described previously (25 (link)). Briefly, tumor tissues were cut into pieces and digested with DMEM containing hyaluronidase (1.5 mg/ml, Sigma-Aldrich, USA), collagenase type 1A (1.5 mg/ml, Sigma-Aldrich, USA), and deoxyribonuclease I (20 U/ml, Sigma-Aldrich, USA) at 37°C for 45 min. The tissue mixture was passed through a 70-µm nylon cell strainer to make single cell suspension, which was then washed and resuspended in cold flow buffer (1% bovine serum albumin and 0.1% NaN₃ in PBS). After being blocked with a rat anti-mouse CD16/CD32 antibody (BD Pharmingen, USA), the following fluorochrome-conjugated anti-mouse antibodies were used: CD45-BV421, CD11b-BV510, CD8a-PE-Cy7, CD4-PE, CD25-APC, NK1.1-APC-Cy7, F4/80-FITC, CD206-PE-Cy7, CD11c-APC, and Gr-1-APC-Cy7 (all from BioLegend, USA). 7-Amino-actinomycin D (7AAD) (eBioscience, USA) was used as a viability dye to exclude dead cells. Flow cytometric data were collected using a Gallios flow cytometer (Beckman, USA) and analyzed using the Kaluza software (version 1.3).

LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit for 405 nm excitation (Invitrogen). AccuCheck Counting Beads (Molecular probes). Cycloheximide (CHX) (Sigma-Aldrich). Phosphoprotein phosphatase (PP1) (Biaffin GmbH). Fluochrome labelled antibodies CD4-FITC (clone SK3), CD8a-PE.Cy7, and HLA-DR-APC.Cy7 (BioLegend), CD3-APC, CD4-PE (clone SK3), CD19-PE, CD14-V450, CD56-FITC, CD64-FITC and Iso IgG1κ-FITC (BD) and CD56-APC (ImmunoTools).

50 µl of blood was 1:1 diluted in PBS; lymphocytes were separated using 100 µl Lymphocyte Separation Medium 1077 (C-44010, PromoCell, Heidelberg, Germany). Cells were stained for 30 min/4 °C with: CD4-PE/Cy5 (1:1000; 130312), CD8a-PE/Cy7 (1:500; 100722), B220-BV510 (1:300; 103248), Gr-1-PE (1:1000; 108408), CD11b-PerCP/Cy5.5 (1:1000; 101227), F4/80-FITC (1:1000; 123107, all from BioLegend). Filtered samples were acquired on FACSAria Sorp (BD-Biosciences), and data analyzed by FlowJo software (BD-Biosciences).

Lung tissue from Balb/c mice injected with 4T1.2 cells was collected and washed using ice-cold PBS. The tissue was minced using forceps and scissors into 1–2 mm sections and pressed through a cell strainer (40 μm cell suspension filter). Whole blood was collected and lysed with cold 1 × RBC lysis buffer (155 mM ammonium chloride, 10 mM sodium bicarbonate, 0.1 mM EDTA) for 10–15 min at room temperature. Lung tissue and whole blood were spun down at 250 g for 10 min at 4°C. The resulting pellet was washed twice with PBS. Subsequently, single-cell suspensions were incubated with Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA) in PBS at 4°C for 30 min. Samples were washed with PBS containing 2% FBS, 2mM EDTA, and 2mM sodium azide, then incubated with antibodies at 4°C for 1 h. Cells were washed with PBS containing 2% FBS, 2 mM EDTA, 2 mM sodium azide, and analyzed using the Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA). Flow cytometry beads (eBioscience, San Diego, CA) stained with each antibody were used as single-color controls. A combination of antibodies was used depending on the purpose of each study: CD45 PE (Biolegend, clone 30-F11), CD3 FITC (Biolegend, clone 17A2), CD8a Pe-Cy7 (Biolegend, clone 53-6.7), CD4 APC (Biolegend, GK1.5).