Anti granzyme b apc

Antibodies (Abs) used for flow cytometry were anti-IFN-γ-PE-Cy7 (BD Pharmingen); anti-CD66b-FITC, anti-CD33-PE, anti-CD45-PE-Cy7, anti-CD14-PerCP-Cy5.5, anti-HLA-DR-Alexa Fluor 647 and CD8-PerCP-Cy5.5 (eBioscience); anti-perforin-PE, and anti-granzyme B-APC (Biolegend). Abs used for western blotting were anti-human arginase I (R&D Systems) and horseradish peroxidase (HRP)-conjugated secondary ab (Zhongshan Biotechnology). Abs used for IL-6/IL-8 blockade studies were: anti-human IL-6 (Biolegend) and anti-human IL-8 (R&D Systems). The chemical arginase I inhibitor nor-NOHA (Santa Cruz) was also used. Purified anti-CD3 and anti-CD28 Abs (Biolegend) and recombinant human IL-2 (PeproTech) were also used for T cell studies.
Recombinant human IL-6, IL-8, and arginase I (Biolegend) were used for CD45⁺CD33^lowCD11b^dim activation studies. Drug inhibitors studies involved the STAT3 inhibitor FLLL32 (Medco Biosciences), PI3K inhibitor Wortmannin, and MAPK inhibitor SB203580 (Calbiochem).

The tumor tissues were collected and cut into small pieces of approximately 1 mm³. The tumor pieces were incubated with a medium containing DNA hydrolase I and collagenase IV for 30 min at 37°C. A 70-μm filter was used to remove non-digested tissue and obtain a single-cell suspension for the following flow cytometry staining. Cells were first stained with Zombie NIR (Fixable Viability kit, BioLegend) to examine live cells and then surface markers staining with the following antibodies at 4°C for 20 min in the dark: anti-CD45-PE (BioLegend, San Diego, CA, USA), anti-CD3-APC (BioLegend), anti-CD4-FITC (BioLegend), anti-CD19-APC (BioLegend), anti-CD11c-FITC (BioLegend), anti-CD86-PerCP/Cyanine5.5 (BioLegend), anti-PD-1-PE/Cyanine7 (BioLegend), anti-PD-L1-PE/Cyanine7 (BioLegend), anti-CD103- PE/Cyanine7 (BioLegend), anti-PNAd-APC (BioLegend). For intracellular markers, cells were fixed and permeabilized using the FoxP3 staining buffer set (eBioscience) and then stained with intracellular antibodies for 30 min at 4°C, including anti-IFN-γ-PerCP/Cyanine5.5 (BioLegend), anti-Granzyme B-APC (BioLegend), anti-Perforin-PE (BioLegend). Flow cytometry analysis was performed using a BD FACSCanto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10 software.