Z aad cmk

The cytotoxicity assay procedure was prepared as mentioned previously. For GZMB inhibition, the effector cells were pre-incubated in IMDM + 10% FBS supplemented with 100 µM Z-AAD-CMK (Enzo Life Sciences; Farmingdale, NY) for 1 h. The effector cells were then added to the wells in a working solution of IMDM,10% FBS and 100 µM Z-AAD-CMK. For GZMA inhibition, the effector cells were pre-incubated in IMDM + 10% FBS supplemented with 5 µM nafamostat mesylate (Selleck Chem; Houston, TX) for 1 h. The effector cells were then added to the wells in a working solution of IMDM,10% FBS and 5 µM nafamostat mesylate.
For antibody blocking, the effector cells were incubated with 10 µg/mL anti-human FasL (CD178) monoclonal antibody (Clone NOK1, BioLegend) for 1.5 h with periodic agitation. The effector cells were then added to the wells in a working solution of IMDM,10% FBS and 10 µg/mL anti-FasL antibody. For IFN-γ and TNF-α blocking, the effector cells were incubated with 5 µg/mL anti-human IFN-γ monoclonal antibody (Clone 4 S.B3, BioLegend) and 5 µg/mL anti-human TNF-α monoclonal antibody (Cline MT25C5, Mabtech; Cincinnati, OH) for 1.5 h with periodic agitation. The effector cells were then added to the wells in a working solution of IMDM,10% FBS and 5 µg/mL anti-IFN-γ and anti-TNF-α antibodies.

Whole blood from healthy donors was collected and neutrophils isolated by negative selection using a direct neutrophil isolation kit (Stem Cell Technologies), yielding purity of >99%. For MRSA killing assays, 4 × 10⁵ neutrophils were incubated in RPMI medium with PBS or IL-21 (100 ng/ml), followed by the addition of 50 μl of MRSA (at a 1:1800 dilution of an optical density at 600 nm (OD₆₀₀) = 0.25) pre-incubated in 10% autologous serum. For granzyme B inhibition, neutrophils were pre-incubated for 20 min with 100 μM Z-AAD-CMK (Enzo Life Sciences). Tubes were rotated at 37°C for 3 hr, and serial dilutions then spread on blood agar plates, incubated overnight, and CFU determined. De-identified whole blood from healthy volunteer donors from the NIH Department of Transfusion Medicine was obtained and met the Office of Human Subjects Research criteria for a waiver for the need for IRB approval.

Universal Type I IFN Alpha (PBL) was used at 5–500 U/mL. Lipofectamine 3000 (Thermo Fisher Scientific) was used to deliver cGAMP (InvivoGen). Z-AAD-CMK (Enzo Life Sciences) was dissolved in water at 10 mM and diluted in medium at a final concentration of 100 μM. Ruxolitinib (LC Laboratories) was dissolved in DMSO at 5 mM and then diluted in medium at a final concentration of 2.5 μM.