Pe conjugated anti mouse cd31

Analysis of apoptotic cells in AGM was performed using the FITC annexin V Apoptosis Detection Kit I (BD Pharmingen, 556547) following the manufacturer’s protocol. Briefly, AGMs from E11.5 embryos were isolated and cultured for 4 days followed by dissociation into single cells and staining with PE-conjugated anti-mouse CD31 (eBioscience, 12-0311-81), APC-conjugated anti-CD117 (c-Kit) (eBioscience, 17-1171-81), and FITC annexin V and PI. Cells were subjected to flow cytometry analysis using BD LSR II. Analysis of cell proliferation in AGM was performed using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen, C10424) following the manufacturer’s protocols. Briefly, three AGMs isolated from E11.5 embryos of the same genotype were mixed and dissociated into single cells and incubated with 10 μM EdU for 1.5 hours and then stained with FITC-conjugated anti-mouse CD31 (BioLegend, 102405). After fixation and permeabilization, cells were processed for Click-iT reaction. Stained cells were labelled with PE-conjugated anti-mouse CD117 and subjected to flow cytometry analysis using BD LSR II.

FBS, DMEM low-glucose medium, DMEM high-glucose medium, RPMI-1640medium, trypsin–EDTA, glutamax and penicillin–streptomycin solution were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant mouse IFNγ and TNFα were purchased from R&D Systems (Minneapolis, MN); Griess reagent were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies used in flow cytometry, including PE-conjugated anti-mouse CD31 (Product code: 11031182), CD34 (Product code: 11034182), Sca-1 (Product code: 12598182), CD44 (Product code: 12044182), CD45 (Product code: 12045182), MHC I KbDb (Product code: 12599882), F4/80 (Product code: 12480182), FITC-conjugated anti-mouse MHC class II I-A (Product code: 12532281), and APC-conjugated platelet-derived growth factor receptor α (PDGFRα) (Product code: 17140181) were purchased from eBioscience (San Diego, CA, USA). Antibodies used in western blotting analysis were: SHP1 (Product code: ab32559), and COX2 (Product code: ab15191) (Abcam, Cambridge, MA, USA), iNOS (Product code: 39898), JAK1 (Product code: 50996), pJAK1 (Product code: 3331), STAT3 (Product code:, pSTAT3 (Product code: 9139), pP38 (Product code: 9216), pJNK (Product code: 9255), pP65 (Product code: 3039), pAKT (Product code: 4060) and GAPDH (Product code: 5174) (Cell Signaling Technology, Danvers, MA, USA).