Cd38 apc hit2

Cell staining was performed on cryopreserved cells that were thawed, washed, and permeabilized with a transcription factor perm/wash buffer (eBioscience, Thermo Fisher Scientific). Cells were then stained at room temperature for 45 minutes with antibodies for CD3 BV650 (OKT3, BioLegend), CD4 BV785 (OKT4, BioLegend), CD8 BV510 (RPA-8, BioLegend), CD19-BV750 (HIB19, BioLegend), CD45RA Alexa Fluor 488 (HI100, BioLegend), CD27 PE-Cy5 (1A4CD27, Beckman Coulter), CD38 APC (HIT2, BD Biosciences), HLA-DR BV605 (L243, BioLegend), Ki67 PerCP-Cy5.5 (B56, BD Biosciences), PD-1 PE (J105, eBioscience, Thermo Fisher Scientific), GrB BV421 (BG11, BD Biosciences), IFN-γ BV711 (4S.B3, BioLegend), TNF-α PE-Cy7 (MAB11, BioLegend), and IL-2 PE/Dazzle 594 (MQ1-17H12, BioLegend). Samples were acquired on a BD Biosciences LSR II and analyzed using FlowJo v9.9.6. A positive response was defined as any cytokine response that was at least twice the background of unstimulated cells. To define the phenotype of SARS-CoV-2–specific CD4⁺ T cells, a cutoff of 20 events was used.

Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies) and then with anti-human CD34 PE-Cy7 (581, BioLegend; 1:100), CD38 Alexa Fluor 647 (AT1, Santa Cruz Biotechnologies; 1;50), CD45RA BV 421 (HI100, BD Biosciences; 1:25), and CD90 BV605 (5E10, BioLegend; 1:30) and analyzed by flow cytometry. For sorting of CD34⁺ or CD34⁺ CD38⁻ CD90⁺ cells, cord-blood-derived CD34⁺ HSPCs were stained directly after isolation from blood with anti-human CD34 FITC (8G12, BD Biosciences; 1:100), CD90 PE (5E10, BD Biosciences; 1:50), CD38 APC (HIT2, BD Bioscience; 1:50), and cells were sorted on a FACS Aria II (BD Bioscience).