Cd45 fitc

Cell media MCDB201, SMEM, DMEM, and RPMI 1640 were obtained from Life Technologies, human antibodies CD14-FITC, CD29-FITC, CD34-FITC, CD44-FITC, CD45-FITC, and CD90-FITC were from GeneTex, CD73-FITC and HLA-DR-PE were from eBioscience, mouse anti-human IgG1-FITC, IgG2a-FITC, and IgG2a-PE were from GeneTex, anti-CD3 and anti-CD28 were from BD Biosciences, Hoechst 33342, Lysotracker Green DND-265, and 6-carboxyfluorescein N-succinimidyl ester (CFSE) were from Invitrogen, Alizarin Red S, Alcian Blue, Oil Red O, phosphate-buffered saline (PBS), human serum albumin (HSA), erythrocyte lysis buffer, and all other chemicals were from Sigma-Aldrich and used without further purification.

Cryopreserved MSCs from QE-1 and QE-2 for Pigs 1 and 3 and passage 3 cells (manually expanded) for Pig 2 were thawed at 37 °C, counted with a hemocytometer, and 1 × 10⁶ cells were transferred to polystyrene tubes for incubation with staining buffer [DPBS supplemented with 0.2% BSA (BD Biosciences)] and the appropriate antibody cocktail for 30 min at room temperature. CD44-APC, CD90-PE (Stem Cell Technologies), and CD105-Alexa405 (Novus Biologicals) were used to identify the MSC populations. CD31-FITC, CD45-FITC, and Swine-Leukocyte-Antigen Class II-FITC (SLA-DRII) (GeneTex) were used to determine the presence of non-MSC lineage positive cells in the expanded MSCs. Prior to acquisition on the BD LSRII flow cytometer, cells were stained for 10 min using 7AAD viability dye (BD Biosciences). Data were analyzed using FlowJo software version 9.9 (FlowJo). A compensation matrix was generated using single color stains on MSCs. Doublets were excluded using FSA vs. FSH gating and debri was excluded using FSA vs. SSC gating for all samples before gating on total live cells negative for 7AAD (BD Biosciences). ‘Fluorescence Minus One (FMO)’ controls were used to identify positive populations.