Cryopreserved PBMC/cord BMC (CBMC) were thawed, counted, and processed immediately for phenotypic assessment using two staining panels. Panel A consisted of surface staining with Zombie yellow (viability), CD25 FITC (BioLegend), Lag3 PE (Invitrogen), CTLA4 PE-CF594 (BD Biosciences), CD4 PerCP-Cy5.5 (BD Biosciences), CD3 Ax700 (BD Biosciences) followed by fixation and permeabilization using the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (eBioscience). Intracellular staining was then performed with FoxP3 Ax647 (BD Biosciences), Granzyme B APC-fire750 (BioLegend), IL-10 BV421 (BioLegend) and TGFβ PE-Cy7 (BioLegend). Panel B consisted of surface staining with Zombie yellow (viability), CD4 FITC (BioLegend), CD3 PE-CF594 (BD Biosciences), GITR PerCP-Cy5.5 (BioLegend), TNFR2 PE-Cy7 (BioLegend), CD39 Ax700 (R&D Systems), PD1 APC-Cy7 (BioLegend) and TIGIT BV421 (BioLegend). Intracellular staining consisted of FoxP3 Ax647 (BD Biosciences) and IL-35 PE (BioLegend). Analysis was performed using the Galios instrument (Beckman Coulter).
Johnson M., Lazarus S.K., Bennett A.E., Tovar-Salazar A., Robertson C.E., Kofonow J.M., Li S., McCollister B., Nunes M.C., Madhi S.A., Frank D.N, & Weinberg A. (2024). Gut Microbiota and Other Factors Associated With Increased Regulatory T Cells in Hiv-exposed Uninfected Infants. Research Square.
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