Anti cd4 vioblue

Freshly isolated CD3⁺CD25⁻ murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5 × 10⁴ BM- or FL-MSCs seeded in 6-well plates 1 day before were then co-cultured with 25 × 10⁵ activated CD3⁺CD25⁻ murine responder T cells (MSC to T cell ratio used 1:5) in a total volume of 3 ml of 50% RPMI-50% MEMα media. 1 × 10⁵ activated and non-activated murine CD3⁺CD25⁻ T cells grown alone in 50% RPMI-50% MEMα medium were used as controls. After either 1 or 3 days, murine T cells were harvested by gentle aspiration and stained with either VIOBLUE-anti-CD4, VioBright FITC-anti-CD8α, PE-anti-GITR, PE-Cy7-anti-CD25 (all from Miltenyi Biotec) and PE-Cy5.5-anti-Foxp3 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-Vio770-anti-ICOS, APC-anti-TNFR2 (all from Miltenyi Biotec), and PE-Cy5.5-anti-Foxp3 (eBioscience) antibodies (Abs).

Freshly isolated CD3⁺CD25⁻ murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5 × 10⁴ BM- or FL-MSCs seeded in 6-well plates 1 day before were then co-cultured with increasing numbers of activated murine CD3⁺CD25⁻ responder T cells (MSC to T cell ratios used 1:1, 1:2, 1:4, 1: 6, 1:8, and 1:10) in a total volume of 3 ml 50% RPMI-50% MEMα medium. 1 × 10⁵ murine activated and non-activated CD3⁺CD25⁻ murine T cells grown alone in culture were used as controls. After 4 days, T cells are harvested and stained using the following Abs: VIOBLUE-anti-CD4, FITC-anti-CD8α, PE-Cy7-anti-CD25, PE-anti-CTLA4, APC-anti-TNFR2 (all from Miltenyi) and Foxp3-PE-Cy5.5 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-anti-GITR, PE-Vio770-anti-ICOS (all from Miltenyi Biotec), and PE-Cy5.5-anti-Foxp3 (eBioscience).

We evaluated the cell viability and phenotype in the different stages of the procedure by flow cytometry. Briefly, cell surface markers staining was followed by staining with Fixable Viability Dye-eFluor450 (eBioscience). Then, the cells were fixed and permeabilized using the FOXP3 transcription factor staining kit (eBioscience) for intracellular staining. All the antibodies are listed in Supplementary Table 2. Flow cytometry analysis of labeled cells was performed with a MACSQuant16 cytometer (Miltenyi Biotec), acquiring at least 100,000 events, and the data were analyzed using Kaluza software (Beckman Coulter).
To isolate the CD4⁺ single-positive (SP) and CD4⁺CD8⁺ double-positive (DP) thyTreg cells, 50x10⁶ of total thyTreg cells were labeled with anti-CD4-VioBlue (Miltenyi Biotec) and anti-CD8-FITC (Beckman Coulter). Cells were washed and resuspended at 5x10⁶ cells/ml in MACSQuant Tyto Running Buffer (Miltenyi Biotec) and were subjected to two consecutive rounds of sorting with High-Speed MACSQuant Tyto Cartridges (MACSQuant Tyto cell sorter, Miltenyi Biotec). After the first round, CD4⁺SP cells were collected from the positive fraction. The negative fraction was loaded into a second cartridge, and CD4⁺CD8⁺ DP cells were collected from the positive fraction.