Macs human cd34 microbead kit

Peripheral blood was first diluted in PBS (1×) and white blood cells were separated from red blood cells and plasma using Ficoll, while leukapheresis bags were directly thawed and diluted in Miltenyi buffer. Then, CD34⁺ HSPCs were isolated using the Miltenyi Biotec MACS human CD34 MicroBead kit (Miltenyi Biotec, San Diego, CA, USA) following the manufacturer’s instructions. With 100 mL of peripheral blood, an average of 1.5 × 10⁶ CD34⁺ HSPCs were obtained. Cells were cultured in complete medium consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with FBS, BSA, glutamine, penicillin/streptomycin, human interleukin (hIL)-3, hIL-6, and human stem cell factor (h-SCF) (PeproTech, Rocky Hill, NJ, USA) at 37°C. Cell proliferation and viability were determined using an automated cell counter (Bio-Rad, Hercules, CA, USA). CFU assays were performed using MethoCult H4434 enriched methylcellulose (STEMCELL Technologies, Cambridge, MA, USA). Two days after electroporation, 3,000 cells from each condition were mixed with 3 mL of MethoCult and plated in triplicate into 35-mm gridded cell culture dishes. After 12–14 days of culture at 37°C, 5% CO₂, the different types of hematopoietic colonies were identified and counted. CFU (∼15/type) were plucked for genomic DNA isolation using QuickExtract (Lucigen, Middleton, WI, USA).