FITC Hamster Anti-Mouse CD40

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol	Cells were acquired on the BD FACSCANTO II and LSR (BD Biosciences) and analysis was carried out using FlowJo (Tree Star).

Protocol tips
Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol
Downstream tips
Cells were acquired on the BD FACSCANTO II and LSR (BD Biosciences) and analysis was carried out using FlowJo (Tree Star).

Publication protocol

Tumors tissues were harvested from mice that had been treated with mAb or otherwise and processed for flow cytometry analysis as previously described (20). For surface staining, tumor-filtrating leukocytes (TIL) were stained with eFluor780 anti-CD45.2 (104; eBioscience), eFluor450 or Brilliant Violet 605 anti-CD4 (RM4-5; eBioscience and Biolegend), PE-Cy7 or Brilliant Violet 421 anti-CD8a (53-6.7; eBioscience and Biolegend), FITC or PE anti-TCRβ (H57-597; eBioscience), PE-Cy7-anti-CD11b (M1/70; eBioscience), eFluor450-anti-Gr1 (RB6-8C5; eBioscience), FITC- or PE-anti-PD1 (J43; eBioscience and BD Pharmingen), APC-anti-PDL1 (10F.9G2; Biolegend), PE-anti-CD27 (LG.3A10; BD Pharmingen), PE-anti-CD86 (GL1; BD Pharmingen), APC-anti-CD80 (16-10A1; eBioscience), PE-anti-CD70 (FR70; BD Pharmingen), FITC-anti-CD40 (HM40-3; BD Pharmingen), Biotin-conjugated-anti-CD28 (37.51; Biolegend), PE-Cy7- or APC-conjugated streptavidin (eBioscience), Alexa Fluor 488-anti-CD25 (PC61.5; eBioscience), Brilliant Violet 605-anti-CD127 (A7R34; Biolegend), PE-Cy7-anti-CD278 (ICOS; 7E.17G9; eBioscience), APC-anti-CD223 (Lag3; C9B7W; Biolegend), PE-anti-CD366 (Tim3; RMT3-23; Biolegend), APC-anti-TIGIT (1G9; Biolegend), PE-Cy7 anti-CD39 (24DMS1; eBioscience), PE-anti-CD73 (TY/23; BD Pharmingen), APC-anti-CD44 (IM7; Biolegend), FITC-anti-CD62L (MEL-14; Biolegend), and respective isotype antibodies in the presence of anti-CD16/32 (2.4G2). 7AAD (Biolegend) or Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol, and stained using eFluor450-anti-Foxp3 (FJK-16s, eBioscience), FITC-anti-Tbet (4B10; Biolegend), APC-anti-CTLA4 (UC10-4B9, eBioscience), Alexa Fluor 647-anti-Ki67 (B56), eFluor660-anti-Eomes (Dan11mag; eBioscience), and respective isotype antibodies. For intracellular staining of IFNγ/TNF or IL12p40, cells were stimulated in vitro with 50 ng/mL PMA (Sigma Aldrich) and 1 μg/mL ionomycin (Sigma Aldrich), or 100 ng/mL LPS, respectively in the presence of GolgiPlug (BD Biosciences) for 4 hours, and then surface stained as aforementioned. Surface-stained cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's protocol, and stained with PE-anti-IL12p40 (C15.6; BD Pharmingen), PE-anti-IFNγ (XMG1.2; eBioscience), Alexa Fluor 647-anti-granzyme B (GB11; BD Pharmingen), and Brilliant Violet 605-anti-TNF (MP6-XT22; Biolegend), and respective isotype antibodies. Cells were acquired on the BD FACSCANTO II and LSR (BD Biosciences) and analysis was carried out using FlowJo (Tree Star).

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