αcd16 32

Flow cytometry analysis was conducted accordingly to the recently published guidelines 45. Organs were mechanically disrupted with scissors and digested for 45 min at 37°C in an enzyme mix composed of: DNase I (0.28 mg/mL, Amresco, Fountain Parkway Solon, OH), and 0.26 units/mL of Liberase TL Research Grade (Roche, Basel, Switzerland)) in RPMI 1640 Medium (Gibco, Bleiswijk, Netherlands) followed by a stop solution of 2 mM EDTA (Sigma–Aldrich, San Luis, MO) and 2% heat‐inactivated filter‐sterilized FCS (Thermo Fisher Scientific, Waltham, MA) in PBS (Sigma–Aldrich, San Luis, MO). Single cell populations were obtained by forcing the remaining tissue pieces through a 40‐µm strainer followed by lysis of RBCs. Fc receptors from the isolated cells were blocked (αCD16/32, Biolegend, San Diego, CA) followed by surface staining and analysis by flow cytometry on a LSRFortessa^TM (BD Biosciences, Franklin Lakes, NJ). Where indicated, intracellular staining was performed according to eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (eBioscience, Santa Clara, CA) following the manufacturer's instructions. Dead cells were excluded using ZombieAcqua fixable viability dye (Biolegend, San Diego, CA) and data were analyzed using FlowJo software (TriStar Inc, Phoenix, AZ). For detection of Vγ6⁺ cells, samples were pre‐stained with GL3 followed by 17D1.

PECs were collected and washed once. For latency experiments, cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). Total PECs were used for MHV68 acute infection experiments. CD11b+ cells or whole PECs were Fc blocked with α-CD16/32 (Biolegend) and stained with Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo) or APC-Cy7-α-CD11b (M1/70, Biolegend), and R718-α-CD102 (3C4 (MIC2/4), BD Biosciences). Cells were counted again and resuspended in 3 x 10⁶ cells/mL before adding CC4F-AM (LiveBLAzer FRET-B/G Loading Kit with CCF4-AM, ThermoFisher Scientific). Cells were incubated for 1 hour, then washed with PBS and fixed with 2% formaldehyde. Samples were immediately run on a Novocyte 3000 (ACEA Biosciences).

Peritoneal exudate cells (PECs) were collected by lavage with 10 mL of flow buffer (1% FBS in PBS (Corning)). PECs were washed once, then Fc receptors were blocked with α-CD16/32 (Biolegend). The following antibodies were used in various experiments: Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), PE-Cy7-α-CD19 (1D3, Tonbo), Violetfluor450-α-CD3 (17A2, Tonbo), and BV711-α-Siglec F (E50-2440, BD Biosciences). Cells were fixed with 2% formaldehyde (VWR). Samples were run on a Novoctye 3000 (ACEA Biosciences) and analyzed with either Flowjo (Tree Star, Inc, San Carols, CA) or NovoExpress (version 1.5.0, Agilent Technologies, Inc.).

Peritoneal exudate cells (PECs) were collected by lavage with 10 mL of flow buffer (1% FBS in PBS (Corning)). PECs were washed once, then Fc receptors were blocked with α-CD16/32 (Biolegend). The following antibodies were used in various experiments: Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), PE-Cy7-α-CD19 (1D3, Tonbo), Violetfluor450-α-CD3 (17A2, Tonbo), and BV711-α-Siglec F (E50-2440, BD Biosciences). Cells were fixed with 2% formaldehyde (VWR). Samples were run on a Novoctye 3000 (ACEA Biosciences) and analyzed with either Flowjo (Tree Star, Inc, San Carols, CA) or NovoExpress (version 1.5.0, Agilent Technologies, Inc.).

Minced tumors were digested with the dissociation buffer containing 1 mg/ml Collagenase B (11,088,807,001, Roche), 0.5 mg/ml DNase I (abs47047435, Absin), and 0.5 mg/ml Hyaluronidase (H3884, Sigma-Aldrich) at 37 °C for 40 min. Then, the suspension was filtered, and the centrifuged cells were treated with red blood cell lysis buffer (C3702, Beyotime). Subsequently, the cells were labeled with Fixable Viability Stain 780 (565,388, BD) and blocked with α-CD16/32 (101,339, BioLegend). Staining reagents used in flow cytometry included Abs targeting CD45 (560,510, BD), CD3e (562,600, BD), CD8α (563,068, BD), CD49b (740,363, BD), CD44 (561,859, BD), CD62L (553,152, BD), CD69 (566,500, BD), CD25 (553,075, BD), Ki67 (556,027, BD), Perforin (11-9392-82, ThermoFisher), Granzyme-B (372,204, BioLegend), TNF-α (563,943, BD), IFN-γ (560,660, BD), CD11c (566,504, BD), CD80 (560,016, BD), CD86 (561,962, BD), CD11b(101,206, BioLegend), F4/80 (565,411, BD), CD206 (141,708, BioLegend), I-A/I-E (107,608, BioLegend). Auxiliary reagents used in flow cytometry included Brilliant Stain Buffer (563,794, BD), GolgiPlug (555,029, BD), and FOXP3 Fix/Perm Buffer Set (421,403, BioLegend). Cell number per 100 mg tumor tissue was measured by Vi-Cell Auto (Beckman). All flow cytometry assays were conducted by FACSCelesta (BD) and analyzed by FlowJo (BD).

PECs were collected and washed once. For latency experiments, cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). Total PECs were used for MHV68 acute infection experiments. CD11b+ cells or whole PECs were Fc blocked with α-CD16/32 (Biolegend) and stained with Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo) or APC-Cy7-α-CD11b (M1/70, Biolegend), and R718-α-CD102 (3C4 (MIC2/4), BD Biosciences). Cells were counted again and resuspended in 3 x 10⁶ cells/mL before adding CC4F-AM (LiveBLAzer FRET-B/G Loading Kit with CCF4-AM, ThermoFisher Scientific). Cells were incubated for 1 hour, then washed with PBS and fixed with 2% formaldehyde. Samples were immediately run on a Novocyte 3000 (ACEA Biosciences).