Anti cd3e apc cy7

Mouse spleens were collected, ground and filtered through a sieve to obtain single cell suspensions. To detect Th1, Treg and Th17, cells were stimulated with phorbol myristate acetate (PMA)/ionomycin mixture and GolgiPlug (BD Biosciences) for 4h. Anti-CD4-FITC (BD Biosciences, 553046), Anti-CD3e APC-Cy7 (BD Biosciences, 557596) and Anti-CD25-BV421 (BD Biosciences, 607180) were used to stain the surface markers. After washing, cells were fixed and permeated using the Fixation/Permeabilization Kits (eBioscience). Anti-Foxp3-Alexa 647 (BD Biosciences, 560401), Anti-IFNG-PE-Cy7 (BD Biosciences, 557649), Anti-IL17A-PE (BD Biosciences, 559502) antibody was used for staining of intracellular markers. Finally, stained cells were detected by flow cytometry (FACSAriaIII, BD Biosciences) and data were analyzed with Flowjo software.

Mouse spleen and Peyer’s patches (PPs) were mashed and passed through 70 μm mesh to prepare a single-cell suspension for FACS analysis. Single-cell suspensions from the spleen were processed by red blood cell lysate buffer and then used for staining. For regulatory T cell quantification, the following antibodies were used: anti CD3e, APC-Cy7 (Cat Number: 557596, BD Bioscience), anti CD4, PerCP-Cy5.5 (Cat Number: 550954, BD Bioscience), anti CD25, FITC (Cat Number: 102006, BioLegend), anti FOXP3, APC (Cat Number: 17-5773-82, eBioscience). Around 1 × 10⁶ cells were seeded into a 96-well plate and stimulated by phorbol myristate acetate (50 ng/ml), ionomycin (1 μg/ml) and golgi inhibitor (1:1000, BD GolgiPlug, Cat Number: 51-2301kz) for Th1/Th2/Th17 cell subset analysis. Then cells were harvested and stained with the following antibodies: anti INF-γ, FITC (Cat Number: 505806, BioLegend), anti-IL-17A, PE (Cat Number: 506904, BioLegend), anti-IL-4, APC (Cat Number: 504105, BioLegend) and anti CD3e, CD4 antibodies as described above. Cells were detected using a BD FACSCalibur flow cytometer (BD Biosciences) and were analyzed using FlowJo software (Version 7.6.1).