Cd4 isolation kit 2

Mouse splenic CD4+ T cells, CD8+ T cells, B cells were isolated using a CD4 isolation kit II (Miltenyi Biotec), CD8a isolation kit II (Miltenyi Biotec) and PanBcell isolation kit II (Miltenyi Biotec) by negative selection, respectively. A purity rate of >96.6% for isolated CD4+ T cells, CD8+ T cells and B cells were confirmed by flow cytometry. Peritoneal macrophages were obtained from B6 and MRL mice by washing their peritoneal cavities with 15 ml of ice-cold saline. The cells from individual mice were centrifuged at 1500 rpm for 5 minutes at 4 °C, washed in complete DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin, and subsequently adjusted to 1 × 10⁷ cells/ml. The cells were cultured on 12-well flat-bottom tissue culture plates (Corning) and incubated for 3 hours at 37 °C under 5% CO₂ air.

CD4+ T cells were isolated from the healthy human peripheral blood by magnetic activated cell sorting (MACS) (negative selection), using the CD4+ isolation Kit II (Miltenyi Biotec, USA) following Ficoll density gradient centrifugation, and then were seeded in 24-well plate (1×10⁶ cells/well) or 96-well plate (1.2×10⁵ cells/well) with the stimulation of ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL, Canada). Then CD4+ T cells were co-cultured with hUC-MSC-sEVs-treated macrophages at a ratio of 40:1 (CD4+ T cells: macrophages). After 5 days, CD4+ T cells were prepared for flow cytometric analysis or Bromodeoxyuridine (BrdU) assay.

Splenocytes and lymphnodes were harvested from 8- to 12-week-old mice and pooled. CD4 cells were MACS (magnetic assisted cell sorting) purified by using CD4 isolation kit II (Miltenyi Biotec, Auburn, CA) or sorted using EasySep kits. Purified CD4 cells were FACS (Fluorescence Activated Cell Sorting) sorted for CD4⁺CD25⁻GFP⁻ T_con cells or CD4⁺CD25⁺ GFP⁺ T_reg cells (>99% purity). In some experiments CD25⁺ T_regs and CD4⁺CD25⁻ CD44^l°^w naïve T_con cells were sorted using EasySep kits, and the purity of T_regs was more than 92%. 6 × 10⁴ FACS sorted T_cons or T_regs were cultured in U-bottom 96 well plates with 2 ng/mL IL-2 only, or also with TLR ligands. Pam3CSK4, heat killed Candida albicans (HKCA), Lipopolysaccharide (LPS), and Flagellin were purchased at Invivogen and were used at following concentrations: LPS, 1 ug/mL; Flagellin, 1μg/mL; Pam3CSK4, 5 μg/mL and HKCA, 10⁶/mL. In some experiments, formaldehyde fixed Candida albicans germtube (FCA) were used at 10⁶/mL.

Mouse CD4⁺ and CD8⁺ T cells and B cells in spleen were isolated using a CD4 isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), CD8a isolation kit II (Miltenyi Biotec), and Pan-B cell isolation kit II (Miltenyi Biotec) by negative selection, respectively. A purity rate of >96.6% for isolated CD4⁺ and CD8⁺ T cells and B cells was confirmed by flow cytometry [43 (link)].

CD4+ T cells, derived from PBMNC of healthy donors by using the CD4 isolation kit II (Miltenyi Biotec, Bergisch Gladbach), were further divided into CD161+ and CD161− T-cell fractions by a staining with an anti-CD161–PE mAb, followed by incubation with an anti-PE microbead mAb (Miltenyi Biotec). Then the two cell subsets CD4+CD161+ and CD4+CD161− were cultured under limiting dilution (0.3 cell/well) in presence of 10⁵ irradiated (9000 rad) allogeneic PBMCs as feeder cells, 1% PHA (vol/vol), and 50 U/ml rIL-2 (Proleukin, Prometheus, Inc. San Diego, USA), in order to obtain T cell clones. Recovered CD4+ T cell clones were classified on the basis of their ability to produce IFN-γ and/or IL-17 and to express surface marker CD161, as previously described [24 (link)]. Briefly, T cells were polyclonally stimulated with PMA plus ionomycin, fixed in formaldehyde and then analyzed for intracellular cytokines production on a BDLSR II flow cytometry (BDBiosciences). Selected T-cell clones of each phenotype were further analysed by flow cytometry for surface expression of CXCR3A and CCR6. Seven Th17, non-classic -Th1 and classic Th1 clones were stimulated at a culture concentration of 10⁶ cells/ml for 72 h in presence of medium or mAbs anti- anti-CD3-CD28 (5 μg/ml each), then cultures supernatants were collected and stored at −30 °C for further analysis.