Anti cd34 ep373y

The following antibodies and reagents were used for the analysis of synovial cells with flow cytometry and cell sorting: anti-CD45-APC-H7 (2D1, BD Pharmingen), anti-CD235a-APC-Alexa Fluor750 (11E4B-7-6(KC16), Beckman Coulter), anti-CD31-PE-Cyanine7 (WM-59, eBioscience), anti-CD146-BV450 (P1H12, BD Horizon), anti-CD34-PE (4H11, eBioscience), anti-PDPN-PerCP-eFluor710 (NZ-1.3, eBioscience), anti-THY1-FITC (5E10, BD Pharmingen), anti-cadherin-11-biotin (23C6), human TruStain FcX (BioLegend), streptavidin-APC (Jackson ImmunoResearch), Live/Dead fixable aqua dead cell stain kits (Molecular Probes). For immunofluorescence staining of synovial tissue, following antibodies and reagents were used: anti-CD45 (135-4C5, AbD Serotec), anti-CD34 (EP373Y, Abcam), anti-PDPN (NZ-1.3, eBioscience), anti-THY1 (F15-42-1, Merck Millipore, and clone Thy-1A1, R&D Systems), anti-cadherin-11-Biotin (23C6), anti-Ki67 (16A8, BioLegend), anti-mouse IgG1-FITC (Southern Biotech), anti-mouse IgG2a FITC (Southern Biotech), anti-mouse IgG2b-Alexa Fluor 647 (Life Technologies), anti-rat IgG-Alexa Fluor 594 (Life Technologies), anti-rat IgG-Alexa Fluor 647, anti-rabbit IgG-Alexa Fluor 546 (Life Technologies), Hoechst 33258 (Life Technologies), and anti-FITC Alexa Fluor 488 (Life Technologies).

To analyze the differentiation efficiency of the coculture system, hESCs/OP9 coculture cells were treated with 0.25% trypsin (Gibco, USA) for 20 min at 37 °C on days 8, 10 and 12. trypsinization was terminated by adding a coculture medium. Co-culture cells were harvested by centrifugation, and washed twice by washing buffer (PBS supplemented by 5% FBS) [16 ]. These cells were immediately incubated with APC-human TRA-1–85 (R&D, USA), PE-Cy5.5 mouse anti-human CD34 (Becton Dickinson Company, USA) for 30 min at 4 °C. After staining, these cells were washed with washing buffer, centrifuged, and resuspended with moderate washing buffer, followed by flow cytometric analysis (BD FACSAria™ II sorter (BD Biosciences, USA). Data were analyzed with FlowJo version 7.6.1 (FlowJo, LLC). For negative control, APC mouse anti-human CD34 (Becton Dickinson Company, USA) was used to HN14, while OP9 cells labeled anti-CD34 [EP373Y] (Abcam, USA) and used Donkey Anti-Rabbit IgG (H&L) (Abcam, USA) as the secondary antibody.

Cell proliferation in the rats was analysed by quantifying the pulmonary cells that incorporated the exogenously supplied 5-ethynyl-2′-deoxyuridine (EdU) to identify the cells undergoing DNA replication. EdU was intraperitoneally injected into the rats at 50 μg/g 24 h before sacrifice [21 (link)]. EdU staining was performed using the Click-iT EdU imaging kit (C10337, Life Technologies) on frozen sections according to the manufacturer’s instructions. To characterize the EdU-positive cells, we performed double-immunostaining with FITC-labelled anti-α-SMA (clone 1A4, Sigma-Aldrich, Lyon, France, 1/100) or with anti-CD34 (EP373Y, Abcam, Amsterdam, 1/200). Images were captured with an LSM 700 confocal microscope (Carl Zeiss, Le Pecq, France). Images were recorded and analysed with ZEN software (Carl Zeiss).