Cd38 pe cy7

PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.