Brilliant violet 421 anti human cd4

CD4+ T cells were negatively selected from thawed cryopreserved PBMCs. Isolated CD4+ T cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of 10 μM for 15 min. 1 X 10⁶ CFSE-stained CD4+ T cells were either left unstimulated, or stimulated for 24 hours with rGal-9 (200nM, and 500nM). After 24 hours, cells were washed and cultured for another 4 days. Cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) and then stained with the following fluorescently-conjugated monoclonal antibodies: Brilliant violet 421 anti-human CD4 (BioLegend, clone: OKT4) and FE/cy7 antihuman CD45RA (BioLegend, clone: HI100). Rainbow beads (Spherotec) were used to standardize instrument settings between runs. Data were acquired on the flow cytometer as above and were analyzed with FlowJo (TreeStar Inc., Ashland, OR).

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).