Easysep human fitc positive selection kit 2

NK cells were isolated from Buffy Coats obtained from healthy donors using the EasySep Human FITC Positive Selection Kit II (Stemcell Technologies) to deplete CD3⁺, CD14⁺, and CD19⁺ cells in the presence of anti-human CD32 (Fc gamma RII) Blocker (Stemcell Technologies). Subsequently, the remaining cells were stained with CD3 FITC (Beckton Dickinson), CD56 PE-Cy7, CD45 Krome Orange (both Beckman Coulter), and LIVE/DEAD Fixable Near-IR Dead Cell fluorescent dye (Invitrogen). Lineage negative and CD56⁺ NK cells were sorted on the FACSaria IIu (Beckton Dickinson), and the purity of NK cells (>95%) was checked during the procedure.

CD34⁺ CD31⁺ endothelial progenitor cells (EPCs) were differentiated from hiPSCs as described.³², ³³ In brief, hiPSCs were seeded onto a Matrigel‐coated plate in mTeSR1 supplemented with 5 μmol/L ROCK inhibitor Y‐27632 for 24 hours (day ‐3). Seeding densities at day ‐3 were optimized between 16,000/cm² and 132, 000/cm² depending on donor and passage in order to obtain high number of CD34⁺ CD31⁺ EPCs. At day 0 and day 1, medium was changed to LaSR basal medium (Advanced DMEM/F12, 2.5 mmol/L GlutaMAX, and 60 μg/mL ascorbic acid) supplemented with 8 μmol/L CHIR99021. At day 2, medium was switched to LaSR basal medium and changed every day for another 3 days. At day 5, CD31⁺ EPCs were purified using FITC‐conjugated human CD31 antibody (Miltenyi Biotec, clone AC128) and EasySep Human FITC Positive Selection Kit II (STEMCELL Technologies) with an EasySep Magnet kit (STEMCELL Technologies). Purified EPCs were seeded onto collagen IV/fibronectin‐coated Transwell filters at a density of 100,000/cm² or collagen IV (10 μg/mL)‐coated culture plates at a density of 10,000‐20,000/cm² in hECSR medium and used for assay or further extended EC culture.