Anti granzyme b alexa647

MALC obtained at day 10 of growth, were co-cultured with NK cells at ratio E:T 0.5:1 or 1:1 and treated or not with mAbs at 10 μl/ml during 4 h in presence of brefeldin A at 10 μl/mL (except of CD69 detection). Cells were then washed, fixed with 2% PFA during 4 min, washed and permeabilized with 1% saponin during 8 min. Cells were stained with anti-CD56-PE (Miltenyi Biotec), anti-CD3-PE/Cy7 (Beckman Coulter, Roissy, France), anti-CD69-PE-Cy5 (BD Biosciences), anti-perforin (PFN)-FITC (BD Biosciences), anti-IFN-γ-Alexa fluor 647 (BD Biosciences), anti-granzyme B-Alexa647 (BD Biosciences) antibodies during 30 min at 4°C in the dark. Cells were analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

Cryopreserved PBMC from patients hospitalized because of COVID-19 severe interstitial pneumonia (severe) and OTD subjects were thawed, washed and rested for 30 min in complete RPMI-1640 medium supplemented with 10% FCS, 2mM L-glutamine and antibiotic antimycotic solution (100 U/ml penicillin, 0.1 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Sigma- Aldrich, St. Louis, MO, USA) (Complete Medium). Subsequently, PBMC were washed and stained for flow cytometric analysis using the following fluorochrome conjugated antibodies: CD8 BV421, CCR7 BV510, PD1 PE-CF594, CD45RA BV650, CD56 BV786, TIM-3 BB515, CD69 PE, CD4 BB700, CD3 APC-H7 (BD Biosciences, San Diego, CA, USA). After fixation and permeabilization (Fixation/Permeabilization Solution Kit; BD Biosciences), cells were stained with anti-Granzyme B Alexa 647 (BD Biosciences). To detect circulating T follicular helper (TFH) cells 1×10⁶ PBMC were stained with the following antibodies: CD3 BV510, CD4 BB700, CD8 BV786, PD1 PE and CXCR5 BV421. The following antibodies were used to identify plasma cells: CD19 BV605, CD27 BB515, CD38 BV421and CD138 BV480. Flow cytometry was performed with a 12-color Celesta (BD Biosciences) instrument and data analyzed with FlowJo 10 software.