Apc cy7 anti tcr β h57 597

Thymocytes were incubated with Percp-anti-7AAD (BBIlife sciences), PE-Cy7-anti-CD44 (IM7; Biolegend), APC-Cy7-anti-TCR-β (H57-597; Biolegend), PB-anti-CD4 (RM4-5; Biolegend), and BV510-anti-CD8 (53-6.7; Biolegend) antibodies at 4°C for 30 min and pulsed at 37°C water bath for 5, 15, and 30 min with PMA (50 ng/ml) and Ionomycin (500 ng/ml). The cells were fixed, permeabilized, and stained with phosphorylated FoxO-1 (pFoxO-1, S256; Cell Signaling technology) for 1 h, and further stained with an AF488-Goat anti-rabbit (Life technology) secondary antibody for 30 min.
In order to analyze the phosphorylated level of STAT3, thymocytes were stimulated with PMA and Ionomycin or α-GalCer. For PMA and Ionomycin stimulation, thymocytes were incubated with PMA (50 ng/ml) and Ionomycin (500 ng/ml) in 37°C water bath for 5, 15, and 30 min. For α-GalCer stimulation, thymocytes (2 × 10⁶) were seeded in a 24-well plate in 1640+10% FBS with the addition of α-GalCer (125 ng/ml) for 72 h. PE-anti-STAT3 phospho (pSTAT3, Tyr705; Biolegend) were analyzed. The cells were fixed, permeabilized, and stained with PE-anti-STAT3 phospho (pSTAT3, Tyr705; Biolegend) for 1 h. All flow cytometry data were collected using a FACS Canto (BD Biosciences) and analyzed with FlowJo software. Mean fluorescent intensity is indicated.

Single cell suspensions were generated from murine tissues of C57/BL6 and BALB/c mice. Afterwards, cells were treated with FcR-blocking reagent (Miltenyi Biotec) for 10 min at 4°C after washing with flow cytometry buffer. To exclude dead cells, samples were washed twice with PBS and incubated with BD Horizon™ Fixable Viability Stain 510 viability dye (for 10 min at RT in the dark). Subsequently surface staining to discriminate immune cell populations with PerCP anti-CD45 (30-F11, BD Biosciences), APC-Cy 7 anti-TCRβ (H57-597, BioLegend), Pe-Cy7 anti-CD11b (M1/70, BD Biosciences), and anti-CD19 (6D5, BioLegend), was performed. The data was acquired using a BD FACS LSR II instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc, version 9.9.6 or 10.4.1).