CD137 (4-1BB) Monoclonal Antibody (17B5), eFluor 450, eBioscience™

Protocol tips

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	The cells were washed twice with PBS, resuspended and incubated in Fixation/Permeabilization buffer (eBioscience) for 30 min at 4 °C.

Protocol tips
The cells were washed twice with PBS, resuspended and incubated in Fixation/Permeabilization buffer (eBioscience) for 30 min at 4 °C.

Publication protocol

The isolated liver metastasis pancreatic tumor infiltrating immune cells of each mouse were enriched for CD8+ T-cells by negative isolation as per the manufacturer’s protocol (Dynabeads Untouched Mouse CD8 cells, Life Technologies). The isolated CD8+ T-cells were incubated with CD3/CD28 stimulation beads (Dynabeads Mouse T-activator CD3/CD28, Life Technologies) according to manufacturer’s protocol at 1:1 cell to bead ratio in T-cell media for 7 h at 37 °C in 5% CO2 before GolgiPlug (BD Biosciences) was added at 1:1000 volume ratio, after which the cells were incubated for 5 more hours at 37 °C in 5% CO2. After a total incubation time of 12 h, the beads were removed as per manufacturer’s protocol and washed 2 times with PBS. The cells from each mouse were kept separate, and were not pooled. The cells were then stained with Live-Dead Aqua (Invitrogen), CD8a PE/Cy7 (clone 53–6.7, Biolegend), PD-1 FITC (clone RMP1–30, eBioscience) and CD137 eFluor450 (clone 17B5, eBioscience) as described above. The cells were washed twice with PBS, resuspended and incubated in Fixation/Permeabilization buffer (eBioscience) for 30 min at 4 °C. The cells were washed twice with Permeabilization buffer (eBioscience), and then incubated with interferon-γ APC (clone XMG1.2, eBioscience) for 30 min on ice. The cells were washed in twice with Permeabilization buffer and resuspended in FACs buffer. IgGκ1 APC isotype control (clone eBRG1, eBioscience) was used for interferon-γ (IFN-γ).


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Manufacturer protocol

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