Alexa fluor 647 conjugated anti foxp3

Fc receptors were blocked with anti-mouse CD16/32 (BioLegend, clone: 93), and surface markers were stained with BV421-conjugated anti-CD3 (BioLegend, clone: 145-2C11), FITC-conjugated anti-CD4 (BioLegend, clone: RM4-5), Alexa Fluor 647-conjugated anti-CD11b (BioLegend, clone: M1/70), PE-conjugated anti-Gr1 (BioLegend, clone: RB6-8C5), PE-conjugated anti-CD44 (BioLegend, clone: IM7), PerCP/Cy5.5-conjugated anti-CD62L (BioLegend, clone: MEL-14), and APC/Cy7-conjugated anti-CD25 (BioLegend, clone: 3C7). After fixing and perminization, intracellular molecules, including cytokines and transcription factors, were stained with PE/Cy7-IL-17A (BioLegend, clone: TC11-18H10.1), PerCP/Cy5.5-conjugated anti-RORγt (BD, clone: Q31-378), Alexa Fluor 647-conjugated anti-FOXP3 (BioLegend, clone:150D), PerCP/Cy5.5-conjugated anti-GM-CSF (BioLegend, clone: MP1-22E9), and Alexa Fluor 647-conjugated anti-TNF-α (BioLegend, clone: MP6-XT22).

Anti-mouse CD16/32 (BioLegend, clone: 93) was used to block Fc receptors, and the surface markers were stained the following antibodies: FITC-conjugated anti-CD4 (BioLegend, clone: RM4-5), BV421-conjugated anti-CD3 (BioLegend, clone: 145-2C11), and APC/Cy7-conjugated anti-CD25 (BioLegend, clone: 3C7). After fix and permeabilization, cytokines and transcription factors were stained with the following antibodies: PerCP/Cy5.5-conjugated anti-RORγt (BD Biosciences, clone: Q31-378), PE/Cy7-IL-17A (BioLegend, clone: TC11-18H10.1), and Alexa Fluor 647-conjugated anti-FOXP3 (BioLegend, clone: 150D).

Spleens and blood from the mice were harvested, and a single cell suspension was prepared. For quantification of the number of Th1/Th2/Th17 cells, cells were stimulated with PMA (50 ng/ml, Sigma), activation cocktail (750 ng/ml, BioLegend), and monensin (2 μmol/L, BioLegend). Cells were thereafter stained with FITC-conjugated anti-CD4 antibodies (BioLegend), permeabilized with permeabilization solution, and then stained with PE/Cy7-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 and APC-conjugated anti-IL-17A (BioLegend). For detection of Treg cells, the cells were stained with PE-conjugated anti-CD25 and Alexa Fluor 647-conjugated anti-Foxp3 (BioLegend). Fluorescence was examined with FACS Canto II (BD Biosciences). All flow cytometry data presented herein were gated by the use of CD4⁺, while IFN-γ⁺ cells represented Th1 cells, IL-4⁺ cells represented Th2 cells, IL-17A⁺ cells represented Th17 cells, and CD25⁺Foxp3⁺ cells represented Treg cells. Data were analyzed with the FlowJo software.