Anti fcr clone 2.4 g2

Tumors were harvested and dissociated into single-cell suspensions. Then, cells were blocked with anti‐FcR (clone 2.4 G2, BD Pharmingen) and labeled with indicated surface markers for 30 min at 4°C. For IFN-γ staining, single-cells were cultured in the presence of a cell activation cocktail (with Brefeldin A) (Biolegend) for 5 h. Cells were permeabilized and stained with intracellular antibodies for 30 min at 4°C as instructed by the manufacturer. Dead cells were excluded using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). The antibodies used in the flow cytometry analysis were: anti-CD45 (clone 30-F11, Invitrogen), anti-CD3 (clone 145–2C11, Biolegend), anti CD4 (clone RM4-5, Biolegend), anti-CD8 (clone 53–6.7, Biolegend), anti-IFN-γ (clone XMG1.2, Biolegend), anti-CD11C (clone N418, Biolegend), anti-CD80 (clone 16–10A1, Biolegend), and anti-CD86 (clone GL1, Invitrogen). Because the specific reaction with ovalbumin-derived peptide SIINFEKL bound to H-2 Kb of MHC class I, anti–H-2kb bound to SIINFEKL (clone 25-D1.16, Biolegend) was used to recognize the tumor specific immune cells. Flow cytometry was performed on the FACS Aria III platform (BD Biosciences, San Jose, CA, USA) and results analyzed using FlowJo software version 10.4 (TreeStar).

The tumors were harvested and dissociated into single‐cell suspensions. The cells were then blocked with anti‐FcR (clone 2.4G2, BD Pharmingen) and labeled with indicated surface markers for 30 min at 4 °C. For IFN‐γ staining, single‐cells were cultured in the presence of a cell activation cocktail (with Brefeldin A; BioLegend) for 5 h. Cells were permeabilized and stained with intracellular antibodies for 30 min at 4 °C as instructed by the manufacturer. Dead cells were excluded using a LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). The antibodies used in the flow cytometry analysis were anti‐CD45 (clone 30‐F11, Invitrogen), anti‐CD3 (clone 145‐2C11, BioLegend), anti‐CD4 (clone RM4‐5, BioLegend), anti‐CD8 (clone 53–6.7, BioLegend), anti‐IFN‐γ (clone XMG1.2, BioLegend), anti‐CD11c (clone N418, BioLegend), anti‐CD80 (clone 16‐10A1, BioLegend), and anti‐CD86 (clone GL1, Invitrogen). Because the specific reaction with the ovalbumin‐derived peptide SIINFEKL bound to H‐2Kb of MHC class I, anti–H‐2 kb bound to SIINFEKL (clone 25‐D1.16, BioLegend) was used to recognize the tumor‐specific immune cells. Flow cytometry was performed on a FACS Aria III platform (BD Biosciences, San Jose, CA, USA), and the results were analyzed using FlowJo software version 10.4 (TreeStar).

To obtain single-cell suspensions, tumor tissues were dissected into approximately 1–3 mm³ fragments and digested by 1 ug/uL collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) and 0.2 ug/uL DNase I (Solarbio, Beijing, China) for 30 min at 37 °C. Cells were blocked with anti-FcR (clone 2.4G2; BD Biosciences, Franklin Lakes, NJ, USA) and then stained with antibodies against CD45, CD3, CD8, CD4, NK1.1, TIGIT, IFN-γ, TNF-α, CD11b, CD11c, I-AK, CD103, IL-10 and IL-12. For intracellular cytokine staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (Cat. 423,303; Biolegend, San Diego, CA, USA) at 37 °C for 4–5 h. After staining of surface markers, cells were fixed and permeabilized followed by staining with IFN-γ, TNF-α, IL-10, and IL-12. Fixable Viability Stain 780 (Cat. 565,388; BD Biosciences) was used for live/dead discrimination. Samples were collected using a FACSCalibur™ flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).