Anti granzyme b qa16a02

The Abs used for cell-surface staining included: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Dazzle 594 (clone 53.6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-Ly-6G PE-Cy7 (clone 1A8), anti-Ly-6C APC-Cy7 (clone AL-21), anti-PD-L1 PE (clone 10F.9G2), anti-PD-1 APC (clone 29F-1A12), CD11b Alexa Fluor 700 (clone M1/70), anti-CD4 Brilliant Violet 570 (clone RM4-5), anti-CD44 PE-Cy7 (clone IM7), CD25 APC (clone 3C7) (Biolegend, San Diego, CA, USA), and CD11c FITC (clone HL3) (BD Biosciences, San José, CA, USA). The abs for intracellular staining were anti-FoxP3 Alexa Fluor 488 (clone MF23) (BD Biosciences) and anti-CTLA-4 PE (clone UC10-4F10-11) (Biolegend). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Thermo Scientific, Eugene, OR, USA) was used for dead cell exclusion. The abs used for intracellular cytokines evaluation were anti-IFNγ Alexa Fluor 700 (clone XMG1.2), TNFα PE-Cy7 (clone MP6-XT22), IL-2 FITC (clone JES6-5H4) (BD Biosciences), anti-perforin (clone S16009A), and anti-granzyme B (QA16A02) (Biolegend).

Multicolor flow cytometry was performed using the following fluorescently conjugated antibodies or reagents: anti-CD3 (OKT3), anti-CD8 (SK1), anti-TCRγδ (B1), anti-GNLY (DH2), anti-CX3CR1 (2A9-1), anti-PDCD1 (EH12.2H7), anti-T-bet (4H10), and anti-Granzyme B (QA16A02), were purchased from BioLegend (San Diego, CA), except anti-DAP12 (406288) (BD Pharmingen™, San Jose, CA) and anti-EOMES (50-4877-42) (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The cells were stained with antibodies for 30 min at 4 °C for surface marker staining. For intracellular marker staining, the cells were permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences), and then stained with the indicated antibodies. The cells were fixed in 0.5% formaldehyde. Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences), and further analyzed using FlowJo (Ashland, OR) Tree Star software.

Seven fluorescently conjugated monoclonal antibodies specific for cell surface proteins were used to analyze NK cells. These comprised anti-CD19 (HIB19; eBioscience/Thermo Fisher Scientific), anti-CD-107a (eBioH4A3; eBioscience/Thermo Fisher Scientific), anti-KIR3DL1 (DX9; BioLegend), anti-NKG2A (REA110; Miltenyi Biotec), anti-LILRB1 (GHI/75; BioLegend), anti-panKIR2D (NKVFS1; Miltneyi), and anti-DNAM1 (11A8; BioLegend). Four fluorescently conjugated monoclonal antibodies were used to detect the following intracellular markers: anti-IFNγ (45.B3; BioLegend), anti-BrdU (51-23619L; BD Pharmingen), anti-granzyme B (QA16A02; BioLegend), and anti-granzyme B (GB11; BD Bioscience).