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).
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Flow cytometry Anti-bodies Mouse - CD274/PD-L1 using APC anti-mouse CD274 (B7-H1, PD-L1) Antibody from BioLegend. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.