Stable Expression of ACVR2B-Fc and EGFP in MSCs

Samples of pPB-CAG-ACVR2B-Fc-His-Puro (5 µg, ACVR2B-Fc) and pPV-EF1a-EGFP-IRES-Puro (5 µg, Control) plasmids were mixed with 1 µg of pHL-EF1a-hcPBase-A plasmid (PBase) in Opti-MEM (Gibco, USA). For IVIS imaging assay, 5 µg of pPV-EF1a-Luciferase-BleoR and 1 µg of PBase plasmids were mixed with 5 µg of pPB-CAG-ACVR2B-Fc-His-Puro and 5 µg of pPV-EF1a-EGFP-IRES-Puro. A total of 100 µl of the DNA and iMSC mixture was obtained. According to the manufacturer's instructions, electroporation was performed using a NEPA21 electroporator (Nepagene, Japan). Stably expressing cells were selected using 1 µg/ml puromycin or 1 µg/ml puromycin (Gibco) with 400 µg/ml zeocin (InvivoGen) for 7 d. FACS analysis for positive MSC markers was performed as previously described [24 (link)].