Pacific blue anti mouse cd4

For surface staining, cells were first incubated with FcγR blocker (CD16/32), followed by fluorochrome-labeled antibodies (Abs). For intracellular staining, cells were stimulated by phorbol myristate acetate (50 ng/ml) and ionomycin (750 ng/ml) for 5 hrs. After incubation, cells were stained for surface markers first, then fixed by using Foxp3/transcription factor staining set followed by intracellular staining. The specific antibodies and their corresponding isotype controls were purchased from Biolegend (San Diego, CA) and eBioscience (Waltham, MA). The following Abs were used in combinations: PE-Cy7 anti-mouse CD3, Pacific Blue anti-mouse CD4, APC-Cy7 anti-mouse CD8, APC anti-mouse CD11b, APC-Cy7 anti-mouse CD11c, AF700 anti-mouse CD19, Percp-cy5.5 anti-mouse CD45R/B220, FITC anti-mouse CD279 (PD-1) and PEAF610 anti-mouse IFN-γ Abs (BD Pharmingen, San Jose, CA). Flow cytometric analysis were done using an LSRII Fortessa (Becton Dickinson, San Jose, CA), and the data analyzed by using FlowJo software 10.0 (TreeStar, Ashland, OR).

For surface staining, cells were first incubated with FcγR blocker (CD16/32), followed by fluorochrome-labeled antibodies (Abs). For intracellular staining, cells were stimulated by phorbol myristate acetate (50 ng/ml) and ionomycin (750 ng/ml) for 5 hrs. After incubation, cells were stained for surface markers first, then fixed by using Foxp3/transcription factor staining set followed by intracellular staining. The specific antibodies and their corresponding isotype controls were purchased from Biolegend (San Diego, CA) and eBioscience (Waltham, MA). The following Abs were used in combinations: PE-Cy7 anti-mouse CD3, Pacific Blue anti-mouse CD4, APC-Cy7 anti-mouse CD8, PE anti-mouse CD44, FITC anti-mouse CD62L, APC anti-mouse CD11b, APC-Cy7 anti-mouse CD11c, AF700 anti-mouse CD19, Percp-cy5.5 anti-mouse CD45R/B220, APC anti-mouse IL-17 Abs, and PE anti-mouse Foxp3 (BD Pharmingen, San Jose, CA). Flow cytometric experiments were performed using an LSRII Fortessa (Becton Dickinson, San Jose, CA), and analyzed by using FlowJo software 10.0 (TreeStar, Ashland, OR).