Pe mouse anti human cd25

To analyze the induction of regulatory T lymphocytes, we cocultured macrophages treated with MSCs without cellular contact in a 0.4-μm Transwell system (Corning) for 6 days in the presence of inducer medium M0, M1 or M2, with CD4⁺ T lymphocytes selected using CD4 MicroBeads (Miltenyi Biotec) in a 1:1 ratio (macrophages:CD4 T lymphocytes) in RPMI medium (HyClone) containing 10% FBS (Biowest) activated with anti-CD2/CD3/CD28 beads (1 bead for each T lymphocyte) (Miltenyi Biotec). After 5 days of coculture, T lymphocytes were collected, washed and blocked with FBS (Biowest) at 4 °C for 15 min. After blocking, FITC mouse anti-human CD4 (BD Biosciences) and PE mouse anti-human CD25 (BD Biosciences) antibodies were added. Then, the cells were stained following the instructions of the FoxP3 staining buffer set (Invitrogen), and PE-Cyanine7 mouse anti-human FoxP3 (eBioscience) was added. Acquisitions were made on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo V10 software. CD4⁺ T lymphocytes cultured in the absence of macrophages were used as a control.

Cells were harvested from plates and centrifuged, and the supernatants removed. PE anti-phospho-Stat3 antibody (8119, CST) and Alexa Fluor® 647 anti-phospho-Stat5 antibody (9365, CST) were used for staining T cells after polarization. Prior to Th17 cell detection, cells were incubated with Brefeldin A (Abcam) for 4-5 h. FITC mouse anti-human CD4 antibody (555346, BD) and PE mouse anti-human IL-17A antibody (560486, BD) were used for staining cells after polarization and treatment. For Treg cell detection, PE mouse anti-human CD25 (555432, BD), and Alexa Fluor 647 Mouse anti-Human Foxp3 (560045, BD) were used for staining cells after polarization and treatment. Flow cytometric detection was performed using BD FACSCanto II. Data were analyzed with FlowJo software (Tree Star Inc.).

The cells were stained with FITC-, phycoerythrin (PE)-, Phycoerythrin-TexasRed (ECD)-, or Phycoerythrin-Cyanin (PC5)-conjugated mAb specific for CD3, CD4, CD8, CD27, CD45RA, CD56, HLA-DR (Beckman Coulter, Marseille, France), CD28, NKG2D (eBioscience, CA, USA) and CCR7 (R&D systems, MN, USA). To determine the regulatory T cell (Treg), we used the following antibodies: PE-Cy™5 mouse anti-human CD4, PE mouse anti-human CD25, and Alexa Fluor® 488 mouse anti-human Foxp3 (BD Biosciences Pharmingen, CA, USA). Cells were incubated at 5°C for 30 min, then washed with phosphate-buffered saline and analyzed by Cytomics FC500 (Beckman Coulter). For Foxp3 detection, cells were permeabilized overnight in Fix/Perm buffer (eBioscience) and then stained with anti-Foxp3. Data acquisition and analysis were conducted with the CXP Software, version 2.2 (Beckman Coulter). The flow data was analyzed by the following method: lymphocyte gates were determined visually in FSC/SSC dot plot after which gates with a positive region of CD3, HLA-DR were determined by staining cells with isotype control antibodies. The others (CD4, CD8, CD28, CD45RA, CD56, CD28, NKG2D and CCR7) were determined visually at a valley point of histogram or dot plot. Tregs phenotype was defined as CD4+ CD25+Foxp3+ cell.