Pe anti human foxp3

Cultured MNCs were collected from each well and centrifuged. The cell pellet was resuspended in PBS. The sample to be tested was incubated with the following antibodies at 4 °C for 30 min. CD45+ lymphocytes, CD3+ T cells, CD3+ CD4+ T cells, and CD3+ CD8+ T cells were detected with the FITC anti-CD3/PE anti-CD8/PerCP anti-CD45/APC anti-CD4 detection kit (ACEA Biosciences, China). CD3− CD56+ CD16+ NK cells and CD3− CD19+ B cells were detected using a FITC anti-CD3/PE anti-CD16+ CD56/PerCP anti-CD45/APC anti-CD19 detection kit (ACEA biosciences). CD4+ CD25+ Foxp3+ Treg cells were detected with FITC anti-human-CD25 (BioLegend), PerCP/Cy5.5 anti-human-CD4 (BioLegend), and PE anti-human-Foxp3 (BioLegend). CD38+ CD3− CD56+ NK cells were detected with PE anti-CD38 (BioLegend), FITC anti-CD3 (BioLegend), and APC anti-CD56 (BioLegend). Lymphocyte subtypes within MNCs were measured with flow cytometry.

Foxp3 is a specific marker of Treg. PBMC were separated from collected blood samples and Treg were detected by the expression of CD25 and Foxp3. We used the following fluorochrome-conjugated antibodies to stain the cell surface antigens: PerCp/Cy5.5 anti-human CD3, Brilliant Violet 421 anti-human CD4, APC anti-human CD25 (BioLegend, San Diego, CA, USA), and Ghost Rad780 Viability dye (Tonbo Bioscience, San Diego, CA, USA). We permeabilized cells using a Human Foxp3 buffer set kit (BD Bioscience, San Diego, CA USA), and stained Foxp3 using PE anti-human Foxp3 (BioLegend, San Diego, CA, USA). FCM was performed using BD FACS CantoII(Ver1.1, Diva6.1, BD Bioscience, San Diego, CA USA) and BD FACSDiva9, FlowJo (BD Bioscience, San Diego, CA USA) was used for data analysis. The gating strategy for the isolation of Treg involved the following steps: (1) isolation of live cells, (2) gating to isolate CD3- and CD4-positive cells, and (3) isolation of CD25- and Foxp3-positive cells (Supplementary Fig. 1).