CD279 (PD-1) Monoclonal Antibody (RMP1-30), FITC, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL.	The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES.	The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter).

Upstream tips
the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL.
Protocol tips
The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES.
Downstream tips
The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter).

Publication protocol

"Following the isolation of liver metastasis pancreatic tumor infiltrating immune cells, the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL. For each treatment group, triplicates of this pooled cell suspension were placed into wells of a 96-well plate at a volume of 100 μL per well. The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES. After Fc blocking, the cells were stained for the following anti-mouse fluorophores for 1 h on ice: CD45 PerCP-Cy5.5 (clone 30-F11, BD Biosciences), CD4 APC-H7 (clone GK1.5, BD Biosciences), CD8a PE/Cy7 (clone 53–6.7, Biolegend), PD-1 FITC (clone RMP1–30, eBioscience), and CD137 APC (clone 17B5, eBioscience) or OX40 APC (clone OX-86, Biolegend).

The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter)."

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