Fitc conjugated mouse igg1

We performed fluorescence-activated cell sorting (FACS) analysis to examine the cell surface expression of TNF-R1 and -R2, using fluorescein isothiocyanate (FITC)-conjugated anti-CD120a (TNF-R1) and anti-CD120b (TNF-R2) human monoclonal antibodies (mAbs; MBL International, Woburn, MA). FITC-conjugated mouse IgG1 was used as an isotype control (BD Biosciences, San Jose, CA). Experiments were performed using a FACS Canto II Flow Cytometer and FACS Diva software (BD Biosciences).

Flow cytometric analysis was performed in P2–P3 expanded BMMSCs and SFMSCs from each donor (n = 5) using a panel of cluster-of-differentiation (CD) antigen antibodies. Briefly, for each staining, 1 × 10⁶ cells were harvested and collected in 1× florescence-activated cell sorting (FACS) buffer (phosphate-buffered saline with 0.1% bovine serum albumin) (Fisher Scientific, Pittsburgh, PA, USA). The Fc receptors were blocked by preincubating cells with 0.5 mg/ml mouse (BD Bioscience, Grand Island, NY, USA) Fc block for 20 min on ice. After repeated washing, cells were incubated with fluorescein isothiocyanate (FITC) preconjugated CD90 (BD Bioscience, Grand Island, NY, USA), CD29 (Beckman Coulter, Brea, CA, USA), CD34 (Millipore, Billerica, MA, USA), major histocompatibility complex II (MHC-II) (AbD Serotec, Raleigh, NC, USA), and non-conjugated CD44 (Abcam, Cambridge, MI, USA) primary antibodies for 60 min in the dark on ice. To detect CD44, cells were stained with the secondary antibody for 30 min on ice (Table 1). Negative control staining was performed using a FITC conjugated mouse IgG1 (BD Bioscience, Grand Island, NY, USA) isotype. The raw data of fluorescence were measured using the BD FACS Diva software (BD Bioscience, Grand Island, NY, USA). The percentages of cells positive for a given protein were recorded.