Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit

Tregs were isolated from PBMC using the EasySep Human CD4⁺CD127^lowCD25⁺ Regulatory T Cell Isolation Kit (Stemcell Technologies). Purity was assessed by flow cytometry and was consistently > 90%. To induce target expression, Tregs were cultured at 37 °C for 48 h in RPMI-1640 with Glutamax, 10% FBS and 10 mM HEPES in the presence of Human T-Activator CD3/CD28 Dynabeads (Gibco) according to instructions of the manufacturer. The expression of CTLA-4 and OX40 was analyzed by flow cytometry.

UCB derived FOXP3⁺ iTregs cells were generated as previous described^{82 (link)}. Ex vivo expansion of FOXP3⁺ iTregs was performed with CD25⁺ cells MACS purified from day 4 TGFβ-induced UCB iTreg. On Day 4 of TGF-β conditioning, CD25 + iTregs were MACS purified, rested for 48 h, and on Day 6, 5 × 10⁵ purified cells were split into expansion cultures with either media containing 100 U/ml IL-2 in suspension or identical media/IL-2 over MSC feeder cells. Cultures were fed every other day with fresh media/IL-2 and MSC during short-term culture (21–28 days). No re-stimulation was performed during the 21–28 day expansion following the initial 4 day stimulation consistent with a previously described UCB Treg expansion protocol^{83 (link)}. For isolation of UCB-derived tTregs, EasySep Human CD4 + CD127lowCD25 + Regulatory T Cell Isolation Kit (Stem Cell Technologies) was used. tTregs were maintained under identical conditions as iTregs. Absolute number of FOXP3⁺ iTreg cells were calculated from the percentage of CD4⁺ cells based on the total viable cell count obtained by trypan blue dye exclusion at each indicated time point. For iTreg stability assays, harvested iTregs were stimulated with CD3 and CD28 antibodies (1 μg ml⁻¹) with IFNγ (10 μg ml⁻¹), IL-6 (10 μg ml⁻¹), and TNF (10 μg ml⁻¹) at concentration of 5 × 10⁵ cells ml⁻¹ for 72 h and analyzed.

Treg (CD4⁺CD25⁺CD127^low) and target cells (CD4⁺CD25⁻ conventional T cells; Tconv) were magnetically isolated from cord blood mononuclear cells using an EasySep™ Human CD4⁺CD127^lowCD25⁺ Regulatory T Cell Isolation Kit (StemCell, Vancouver, BC, Canada). Obtained Treg were used for DNA/RNA extraction and for assessing Treg cytokine production and capacity for suppressing production of selected effector cytokines in a functional assay based on coculture of Treg with Tconv.

Primary CD25-CD4+ effector T cells were isolated from fresh Human Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy donors, after institutional review board–approved informed written consent (STEMCELL Technologies). Peripheral blood mononuclear cells (PBMCs) were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA). The washed PBMCs were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Cells were seeded at 1×10⁶ cells/mL in complete RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887–100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641) and stimulated with 6.25 uL/mL ImmunoCult^™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990). Following activation and electroporation, cells were split 1:2 every other day to maintain an approximate density of 1×10⁶ cells/mL.