Anti human cd45 apc h7

Cells were isolated by the Ficoll-Paque method from peripheral blood (PB) and bone marrow (BM) aspirates. Cells were labeled with anti-human CD45 APC-H7 (BD Biosciences, clone 2D1, cat. 560178), anti-human CD3 PE (BioLegend, clone SK7, cat. 344806) and anti-human CD34 APC (eBioscience, Clone 4H11, cat. 17-0349-42). Cells were washed and resuspended in 4′,6-diamidino-2-phenylindole (DAPI) to exclude dead cells. Populations were sorted using a BD FACSAria-II (BD Biosciences). Total RNA and genomic DNA were extracted from bulk samples and sorted cells with AllPrep DNA/RNA Mini Kit (Qiagen, cat. 80204).

Signal transducer and activator of transcription 5 (STAT5) phosphorylation after stimulation with supernatants containing hIL-7-Fc or not was assessed on whole blood of patients with septic shock and COVID-19 and HVs. Whole blood of patients was stimulated for 10 min using diluted supernatants from MVA-hIL-7-Fc– or empty MVA–infected or –uninfected cells or directly with the rhIL-7. Whole blood cells were stained with anti-human CD3 PE-Cy7 (Clone SK7, BD Biosciences) and anti-human CD45 APC-H7 (Clone 2D1, BD Biosciences). Fixation, red blood cell lysis, and permeabilization steps were performed using Perfix-Expose kit (Beckman Coulter) as recommended by the manufacturer. After permeabilization, cells were stained with anti-human pSTAT5 (Clone 47/Stat5 (pY694), BD Biosciences). Canto II flow cytometer was used to analyze cells and data were analyzed using the FlowJo software.