Live dead amcyan

Naïve CD4⁺ T cells were isolated from spleen and lymph nodes of C57BL/6 mice using naïve CD4 negative selection kit (StemCell) and activated under Th17-polarizing conditions in the presence of irradiated wild-type splenocytes at a 5:1 ratio, with anti-CD3 (2.5 μg/ml) (145-2C11, BioXCell), anti-IL-4 (10 μg/ml) (11B11, BioXCell), anti-IFNγ (10 μg/ml) (XMG1.2, BioXCell), mIL-6 (30 ng/ml) (Miltenyi Biotec) and hTGF-β1 (3 ng/ml) (StemCell). The following day, cells were treated with either DMSO or of FGIN-1-27 (10 μM). For phospho-ZAP70 analyses, cells were stained after 24 hours of treatment using 10 μg/ml of biotinylated anti-CD3 (145-2C11, BD Biosciences) and anti-CD28 (37.51, BD Biosciences) antibodies on ice for 15 minutes, washed, and stimulated in presence of 20 μg/ml of Streptavidin for 10 minutes at 37 degrees. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho ZAP70 antibody (n3kobu5, eBioscience) and anti-CD4 (RM4–5, eBioscience). For phospho-STAT3 analyses, cells were cultured for additional 24 hours in IL-6 free media, before stimulation with 100 ng/ml of mIL-6 for 30 minutes. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho-STAT3 antibody (4/P-STAT3, BD Biosciences) and anti-CD4. Dead cells were excluded using Live/Dead Amcyan (Thermo Fisher) staining prior to stimulation.

Resting NK cells (2 × 10⁵ cells/mL) and mDCs (1 × 10⁶ cells/mL) were co-cultured at a ratio of 1:5 in the 96-well U-bottomed plate for 2 d. The cultured cells were then harvested, and the supernatant was stored for cytokine ELISA. Then, the cells were resuspended in FACS buffer, which was stained with different surface markers, including Live/Dead-Amcyan (Thermo Fisher Scientific), CD49b-PE (BD Pharmingen^TM), CD69-AF700 (activation marker for NK cells; BD Pharmingen^TM), CD11c-PE/Cy7 (Thermo Fisher Scientific), CD40-BV605 (Thermo Fisher Scientific), and intracellular cytokine marker IFN-γ-APC/Cy7 (BD Pharmingen^TM). The cell phenotypes and the frequency of IFN-γ were determined using the Becton-Dickenson LSR flow cytometer and analyzed using the Flowjo Software Program (Tree Star Inc., OR, USA).

Immature DCs (iDCs, 2 × 10⁵ cells/mL) and ANK cells (4 × 10⁵ cells/mL) were co-cultured at a ratio of 1:2 in the 96-well U-bottomed plate. After 2 d of incubation, the cultured cells were harvested. The supernatant was stored for cytokine ELISA and the cells were resuspended in FACS buffer and stained with different surface markers, including Live/Dead-Amcyan (Thermo Fisher Scientific), CD49b-PE (BD Pharmingen^TM), CD69-AF700 (BD Pharmingen^TM, Franklin Lakes, NJ, USA), CD11c-PE/Cy7 (Monoclonal Antibody N418; Thermo Fisher Scientific, Waltham, MA, USA), CD40-BV605 (clone 3/23; BD Biosciences, Franklin Lakes, NJ, USA), and CD86-FITC (clone GL1; BD Pharmingen^TM). Flow cytometry data were acquired using a Becton-Dickenson LSR flow cytometer and analyzed using the Flowjo Software Program (Tree Star Inc., Ashland, OR, USA).