Percp cy5.5 conjugated anti cd90

CD34⁺ cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO₂. Then the cells were collected, respectively, and stained with APC‐conjugated anti‐CD34 (BD; 555824), PE.Cy7‐conjugated anti‐CD38 (BD; 560677), APC.Cy7‐conjugated anti‐CD45RA (BD; 560674), PerCP.Cy5.5‐conjugated anti‐CD90 (BD; 561557) and PE‐conjugated anti‐CD49f (BD; 555736) antibodies for 30 minutes at room temperature in dark. Then 50 CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells were sorted into a mixture of CellsDirect 2× Reaction Mix, 0.2× TaqMan Assay Mix (Applied Biosystems) and SuperScript III RT/ PlatinumTaq Mix (Invitrogen). Total RNA was extracted with CellsDirect One‐Step qRT‐PCR Kit (Invitrogen) according to the manufacturer's instructions. Reverse transcription and specific target amplification were performed continuously with the following parameters: 50°C for 15 minutes, 95°C for 2 minutes, 95°C for15 s for 18 cycles and 60°C for 4 minutes. Pre‐amplified cDNA was diluted with TE buffer (1:5), and PCR assay was performed. Data were analysed using BioMark Real‐Time PCR Analysis Software (Fluidigm).

A total of 1.0 × 10⁶ passage 3 cells were incubated per tube with PerCP Cy5.5-conjugated anti-CD90, APC-conjugated anti-CD73, FITC-conjugated anti-CD105, and PE-conjugated anti-CD146 antibodies (all from BD Biosciences, Franklin Lakes, NJ) for 30 minutes, at room temperature, and protected from light. Data were acquired using a BD Accuri flow cytometer (BD Biosciences) and analyzed with the CSampler Accuri Software (BD Biosciences).