Phycoerythrin cy7

TILs, NILs, and PBMCs were analyzed by FACS. Cell density was adjusted to 2 × 10⁶/mL. Cells were stimulated by addition of 50 ng/mL phorbol myristate acetate (PMA), 1 μg/mL ionomycin and 10 μg/mL Brefeldin A (BFA) to the medium for 5 h at 37°C and 5% CO2. Then, anti-human-specific antibodies conjugated with fluorescent molecules were added sequentially to the cells. These human antibodies included anti-CD4, anti-IL-17A, anti-CD25, and anti-FOXP3. These were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin, allophycocyanin, or phycoerythrin-Cy7 (BD Biosciences, San Jose, CA, USA). Stained cells were analyzed on an FC500 flow cytometer (Beckman Coulter, CA, USA). CD4+IL-17A+ cells were defined as Th17 cells, whereas CD4+CD25+FOXP3+ cells were defined as Tregs.

Bone marrow MSCs were isolated from male Sprague-Dawley (SD) rats as previously described [18 (link)]. Briefly, bone marrow cells were flushed out with basic culture medium from the femur and tibia bones and seeded in culture dishes containing Dulbecco's modified Eagle's medium : nutrient mixture F-12 (DMEM : F12, HyClone), supplemented by 10% fetal bovine serum (FBS) (Biological Industries) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Sigma). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 48 hours, the dishes were washed with fresh culture medium to remove the nonadherent cells. Cells were harvested with 0.25% trypsin-EDTA (Sigma) and passaged at 5 × 10³ cells/cm². The MSCs (passage 3 [P3]) were identified by flow cytometry using antibodies against CD90-APC (Allophycocyanin, BD Pharmingen), CD45-PE-Cy7 (Phycoerythrin-Cy7, BD Pharmingen), and CD11b-FITC (Fluorescin isothiocyanate, Biolegend). MSCs at P3, P4, and P5 were used for the experiments.