Anti cd34 pe

The enumeration of HSPC was conducted in whole UCB using BD Trucount tubes (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instruction. 100 µl whole UCB were added by reverse pipetting to BD Trucount tubes and stained with anti- CD45− FITC, anti- CD34− PE, anti- CD38− PE- Cy7, anti- CD10− PE- CF594, 7-AAD (all BD Biosciences) and anti- CD133– APC (Miltenyi Biotechnology, Bergisch, Gladbach, Germany). After immunofluorescence staining, erythrocytes were lysed with ammonium chloride lysis solution and analyzed within one hour by flow cytometry (LSRII, BD Biosciences) and analysed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For enumeration of HSPC in UCB, CD34+ cells were gated according to the modified ISHAGE criteria (CD45^dim/7-AAD-/CD34+ cells). CD10 was added to exclude B- lymphoid progenitors. The number of cells/µl was calculated as [(# gated cells/# acquired beads) * (# of beads per test/test volume).

SVF cells were isolated from fat pads and SVF-gel by collagenase digestion. Briefly, SVF-gel and fat pads were digested with phosphate-buffered saline (PBS) containing 0.075% collagenase for 30 min on a shaker at 37 °C. Mature adipocytes and connective tissue were removed by centrifugation at 800g for 5 min. The cell pellet was resuspended and filtered through a 100-μm mesh. The number of SVF cells was counted. SVF cells prepared from SVF-gel were used for flow cytometric analysis and culture of ASCs, while SVF cells prepared from fat pads were used to treat scars.
SVF cells prepared from SVF-gel were characterized by fluorescence-activated cell sorting analysis. The cells were neutralized, washed, filtered, counted, and labeled with the following dyes, antibodies, and corresponding isotype controls for 30 min: 7-AAD (BD Pharmingen, Franklin Lakes, NJ, USA), anti-CD31-Pacific Blue (BioLegend, San Diego, CA, USA), anti-CD34-PE (BD Pharmingen), and anti-CD45-FITC (Thermo Fisher, Waltham, MA, USA). SVF cells were analyzed using an LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA) and counted using an AMQAF 1000 device (Thermo Fisher Scientific, Fremont, CA, USA). Gates were set for isotype controls to exclude > 99.9% of non-specifically stained cells for classification of the cell populations.

Cells were stained with anti-CD31-PE, anti-CD33-FITC, anti-CD34-PE, anti-CD45-FTIC, anti-CD13-PE, anti-CD44-PE, anti-CD45-FITC, anti-CD71-PE, anti-CD90-PE, anti-CD95-APC, anti-HLA-ABC-FITC, anti-HLA-DR-FITC (all from BD Bioscience), anti-CD31-APC (eBioscience), anti-CD13-PE (Biolegend) and anti-CD105-FITC (R&D Systems). The stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).