Easysep human na ve cd4 t cell isolation kit 2

The CD4⁺ naïve T cells were isolated from PBMC on the same day of device assembly (see details below). In brief, PBMCs were thawed in a 37° C water bath for approximately 1 minute before diluting 1:10 in PBS-HIFBS. After that dilution in the isolation buffer, centrifugation at 300 g for 5 min and 4°C was performed, and the isolation buffer was removed from the cell pellet. The cell pellet was then resuspended in 1 mL of PBS-HIFBS and transferred into a 5-ml round-bottom polystyrene tube (StemCell, 100-0088). An additional 1.5 mL PBS-HIFBS was used to recover the residual cells and transferred into the same polystyrene tube. Naïve CD4+ T cells were isolated using the EasySep^™ Human Naïve CD4⁺ T Cell Isolation Kit II (StemCell, 17555) and EasySep^™ Magnet (StemCell, 18000). Once isolated, the naïve CD4⁺ T cells were pelleted and resuspended in 1 mL of RPMI 1640 supplemented with 10% HIFBS (RPMI-HIFBS) and ready for use. Cells were counted via Trypan Blue and Countess II Automated Cell Counter, and 60,000 naïve CD4⁺ T cells were used in each well in circulation.

The subpopulation of CD4+ T lymphocytes was purified by immunomagnetic negative isolation using the EasySep™ human naïve CD4+ T Cell Isolation Kit II (Stem cell, Canadian). The naïve CD4+ T cells were seeded at density of 3×10⁵ cells/well in a 96-well plate. The cells were then transfected either with mock control or with miR-30e-5p mimic or inhibitor obtained from RiboBio (Guangzhou, China) using Entranster™-R4000 Transfection reagent from Engreen (Beijing, China). The cells were then harvested and activated by plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) antibodies, with the addition of recombinant human TGF-β (5 ng/ml) and IL-2 (5 ng/ml) to induce Treg cell conversion or human IL4 (10 μg/ml), IFN-γ (10 μg/ml), TGF-β (2.5 ng/ml), IL-6 (10 ng/ml), and IL-23 (10 ng/ml) to induce Th17 cell conversion. In some cultures, RvD1 (10 nM) was added once a day for 4 days. Cells were collected for measurement of Treg or Th17 cells on the fifth day.

Human donor buffy coats were purchased from Stanford Blood Center, and PBMCs were isolated by centrifugation in Lymphoprep™ density gradient medium (Stemcell Technologies Cat# 07811). The plasma layer was discarded, the white mononuclear cell layer was decanted and washed twice with cold PBS + 2% FBS, and cell count and viability were determined.
For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center. Rested naïve CD4+ T-cells or CD8+ T-cells were prepared by overnight incubation at 37°C, 5% CO₂ in CTS™ OpTmizer™ T-Cell Expansion SFM (ThermoFisher Scientific Cat# A1048501).