Fitc conjugated mouse anti human cd45

Flow cytometry was used to analyze the expression of cell surface markers in RFP- and Oct4/Sox2-ATMSCs. By passage 5, a homogenous population of rapidly dividing cells with fibroblastoid morphology was obtained. ATMSCs were fixed with 70% ethanol at 4°C and stained for 30 min on ice with primary antibodies that recognized various surface molecules. Phycoerythrin-conjugated (PE) mouse anti-human CD29 (BD Bioscience, San Jose, CA), fluorescein isothiocyanate-conjugated (FITC) mouse anti-human CD31 (BD Bioscience), PE-conjugated mouse antihuman CD34 (BD Bioscience), FITC-conjugated mouse anti-human CD44 (BD Bioscience), FITC-conjugated mouse anti-human CD45 (BD Bioscience), PE-conjugated mouse anti-human CD73 (BD Biosciences Pharmingen, San Diego, CA), PE-conjugated mouse anti human CD90 (BD Biosciences), and PE-conjugated antihuman CD105/endoglin (R&D System, Minneapolis, MN) were used for the detection of cell surface antigens. The immunophenotype of MSCs was analyzed with the FACSCalibur flow cytometer (BD Biosciences, Bedford, MA) using the CELLQuest software (BD Biosciences).

For phenotypical identification of AF-MSCs, at least 1 × 10⁵ cells for one assay were collected by centrifugation at 600 ×g for 5 min. Pelleted cells were washed twice in phosphate-buffered saline (PBS) supplemented with 0.2% foetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY, USA). Then cells were suspended in 50 μL PBS with 1% bovine serum albumin (BSA) and incubated with the following antibodies against cell surface markers: FITC conjugated mouse anti-human CD45 (BD Pharmingen, San Jose, CA, USA), CD34 (Miltenyi Biotec, Teterow, Germany), and CD90 (Molecular Probes, Life Technologies, Waltham, MA, USA) or PE labeled mouse anti-human CD105 (Invitrogen, Life Technologies, Waltham, MA, USA). Mouse IgG2A-FITC (Miltenyi Biotec, Teterow, Germany), IgG1-FITC (Invitrogen, Life Technologies, Waltham, MA, USA), or IgG1-PE (Molecular Probes, Life Technologies, Waltham, MA, USA) was used as isotype controls. Samples were incubated in the dark at 4°C for 30 min and washed twice with PBS with 1% BSA. Finally, cells were suspended in 200 μL PBS with 1% BSA and analyzed using Guava easyCyte 8HT flow cytometer (Millipore, USA) with InCyte 2.2.2 software.