Anti cd31 fitc

The phenotype of EA.hy926 were confirmed by immunocytochemical staining for factor VIII and surface expression of platelet/endothelial cell adhesion molecule 1 (CD31) was detected. Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and then incubated with 3% hydrogen peroxide for 20 min at room temperature (RT) to quench endogenous peroxidase activity. Fixed cells were then blocked with 5% bovine serum albumin (BSA) for 20 min and incubated overnight at 4°C with an antibody against factor VIII-related antigen (Boster, China). Immunolabeled cells were washed thoroughly in phosphate-buffered saline (PBS), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Abcam, UK) for 1 h at RT. Immunostaining was visualized by incubation in the chromogen diaminobenzidine (DAB) for 5 min at RT. Expression of CD31 was assessed by flow cytometry. Cells were collected and stained with anti-CD31-FITC (Becton Dickinson, US) for 30 min at RT, followed by fixation for 20 min at 4°C. Samples were loaded in a FACSCalibur system (Becton-Dickinson, US) and results analyzed using FlowJo software (TreeStar, San Carlos, CA, USA).

The day before irradiation, cells were placed in 24-well plates at density 4 × 10⁵ cancer cells per well (monocultures) or 2 × 10⁵ cancer cells and 2 × 10⁵ normal cells per well (co-cultures). Three days after irradiation, cells were detached by 0.25% trypsin and washed with PBS^-. Cells were fixed and permeabilized with 70% ice-cold ethanol and stored at −20 °C until further analysis. Next, cells were washed with PBS and incubated with anti-CD90 FITC (co-cultures of FaDu with Wi-38 cells (BioLegend, San Diego, CA, USA), anti-CD10 APC (co-cultures of A253 with Wi-38, BioLegend) and anti-CD31 FITC (co-cultures with HUVECs, BD Biosciences, San Jose, CA, USA) for 30 min in dark. Then, cells were incubated in PBS^- containing RNase A (1 mg/mL, (Qiagen, Venlo, Netherlands)) and propidium iodide (PI, 1 mg/mL, (Cayman Chemical, MI, USA)) for 1 h at 37 °C. The DNA content of cancer cells was analyzed using BD FACSCanto™ II flow cytometer.

Cells from dissociated EB were filtered through 50 μm filter (Partec) before stained for FACS analysis. All antibodies were purchased from BD Bioscience, including anti-CD34-APC (#555824), anti-CD43⁻FITC (#555475), anti-CD31-PE (#555446), anti-CD31-FITC (#555445), anti-CD73-PE (#550257), anti-CD184- PE-Cy7 (#560669), and anti-CD309- Alexa Flour 647 (#560495). Cells were stained for 30 minutes at 4 degree in the dark then washed twice with PBS supplemented with 2% FBS before flow cytometry. DAPI was used for selection of live cells. All antibodies for flow cytometry were diluted at 1:5 ratio.

Cells were fixed in 4% formaldehyde for 30 min, permeabilized in 0.4% Triton X-100 in PBS for 30 min and incubated with blocking buffer (10% donkey serum in PBS) for 30 min. Samples were incubated with primary antibody overnight in 4 °C and with secondary antibody for 1 h at room temperature. Cell images were taken using confocal microscopy. Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570). Antibodies used were as follows: anti-NANOG (Abcam, ab21624, 1:200), anti-SOX2 (Santa Cruz, sc-17320, 1:100), anti-OCT4 (Santa Cruz, sc-5279, 1:100), anti-TUJ1 (Sigma, T2200, 1:100), anti-αSMA (Sigma, A5228, 1:100), anti-FOXA2 (Cell Signaling Technology, 8186S, 1:100), anti-CD31-FITC (BD Biosciences, 555445, 1:50), anti-vWF (Dako, A0082, 1:200), Dil-Ac-LDL (Molecular probes, 1:400), anti-SM22 (Abcam, ab14106, 1:200), anti-Calponin (BD Biosciences, 2017-03, 1:200) and anti-Ki67 (Vector Labs, VP-RM04, 1:1,000) anti-RelA (Cell Signaling Technology, 8242, 1:200).

To confirm their identities as MSCs, both SHED and OOMDSCs were characterized by flow cytometry for the expression of typical surface markers of MSCs (CD29, CD44, CD90, CD73, CD105, and CD106) and endothelial (CD31) and hematopoietic (C34) cells. All cells were incubated with antibodies (conjugated with fluorochromes) that had the ability to bind specifically to intracellular and cell surface proteins to compare and characterize the cells according to the expression of specific antigens. A total of 1 × 10⁶ cells obtained from cell cultures and diluted in 100 μL of 1XPBS⁻ were transferred to flow cytometry tubes and incubated with the following monoclonal antibodies for 15 minutes at room temperature in the dark for staining: anti-CD29-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, anti-CD105-PE, anti-CD166-PE, anti-CD34-FITC, and anti-CD31-FITC (BD Bioscience, Becton Dickinson Franklin Lakes, NJ). The samples were then washed with 1XPBS⁻, resuspended in 500 μL of 1XPBS⁻, run on a FACSCalibur (BD, Becton Dickinson, Franklin Lakes, NJ) flow cytometer, and subsequently analyzed using FlowJo software (TreeStar Inc.). A sample of unstained cells was prepared for each experiment to eliminate the influence of any nonspecific staining and innate autofluorescence of the cells. As a negative control for the reactions, an isotype control was used for each antibody.

Cells were detached with 0.05% trypsin-EDTA and blocked with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FCS; Gibco BRL), 3.3 mg/mL human immunoglobulin (Gammanorm; Octapharma, Stockholm, Sweden) and 0.1% sodium azide. Cells were labelled with different combinations of the following direct conjugated antibodies: anti-CD105-FITC, anti-CD34-FITC, anti-HLA-DR-FITC, anti-CD31-FITC, anti-CD73-PE, anti-CD14-PE, anti-19-PE, anti-HLAclass I-PE, anti-CD146-PE, anti-CD90-APC, anti-CD45-APC (all from BD Bioscience) and anti-CD271-APC (Miltenyi Biotec). Corresponding isotype controls were all from Becton Dickinson. Before analysis, cells were stained with 7-amino-actinomycin D (1 μL/mL; Sigma Aldrich) for dead cell exclusion. Samples were analysed on an FACSCalibur (BD Bioscience). Data acquisition and analysis were performed using CellQuest (BD Bioscience) and FlowJo software (Tree star, Ashland, Oregon, USA).