Anti human cd31 fitc

ASPCs were cultured in 10 cm dishes until they reached 90% confluence. Cells were released from the plate using Tryple (Invitrogen) and counted followed by a wash with PBS and heat inactivated FBS (FACS buffer). Cells were resuspended to a concentration of 1 × 10^{^6} cells/mL in FACS buffer and 50,000 cells were transferred to each well of a 96-well plate. Cells were stained for surface markers using the following antibodies: anti-human CD31-FITC (BD Biosciences Catalogue number: 557508), CD90-PerCP-Cy5.5 (BD Bioscience, Catalogue number: 561557), CD166-PE (BD Bioscience, Catalogue number: 559263) and CD56-BV605 (BD Bioscience, Catalogue number: 562779). Prior to the final experiment, a titration was performed to identify the optimal antibody dilution. Antibodies were combined as indicated in the figures with a final dilution of 1:50. 100 μl of the antibody mix (containing 2ul of each antibody diluted into FACS buffer) was added to each well and incubated for 20 min. Cells were washed and resuspended in wash buffer (PBS containing 2% heat inactivated FBS and 0.5 mM EDTA). A compensation plate was prepared with positive anti-mouse IgG beads together with individual antibodies for single staining including unstained cells and beads. Data was analyzed using FCS Express 7 Flow version 7.04.0014.

5 × 10⁵ cells were collected and stained with anti-human CD31-FITC (BD biosciences, 557508, 1:100) and anti-human CD144-PE (BD Biosciences 561714, 1:200) and analyzed by a flow cytometry (BD FACSAria IIIu). IgG-FITC (BD biosciences, 555748) and IgG-PE (BD biosciences, 555749) were used as isotype controls.

Cells seeded on microscope coverslips were fixed with 4% formaldehyde for 20–30 min, permeabilized with 0.4% Triton X-100 in PBS for 10–20 min, and blocked with 10% donkey serum in PBS for 1 h at room temperature. Cells were then incubated with primary antibody (diluted with 1% donkey serum in PBS) overnight at 4 °C and fluorescence-labeled secondary antibody (Invitrogen; 1:500 diluted with 1% donkey serum in PBS) at room temperature for 1 h the next day. Hoechst 33342 (Invitrogen; 1:1,000) was used to stain nuclear DNA.
Primary antibodies for immunofluorescence include anti-NANOG (Abcam, ab21624; 1:100), anti-SOX2 (Santa Cruz, sc-17320; 1:200), anti-OCT4 (Santa Cruz, sc-365509; 1:200), anti-TUJ1 (Sigma, T2200; 1:500), anti-α-SMA (Sigma, A5228; 1:200), anti-FOXA2 (CST, 8186S; 1:200), anti-Vimentin (Abcam, ab8978; 1:250), anti-α-Tubulin (Sigma, T5168; 1:500), anti-Vinculin (Sigma, V9131; 1:100), anti-ZOI (Abcam, ab96587; 1:200), anti-ClaudinV (Abcam, ab15106; 1:200), anti-SM22 (Abcam, ab14106; 1:200), anti-Calponin (Dako, M3556; 1:200), anti-vWF (Dako, A0082; 1:200), anti-eNOS (BD, 610296; 1:100), anti-VE-cadherin (CD144) (CST, 2158S; 1:100), anti-human CD31-FITC (BD biosciences, 557508, 1:100), anti-Ki67 (Vector Laboratories, ZA0731; 1:500), Phalloidin (F-actin) (Invitrogen, A22287; 1:50), anti-NF-κB P65 (RelA) (CST, 8242S; 1:200).

Human umbilical cord‐derived MSC were isolated according to the previously described protocol. After receiving the appropriate written consent, MSC harvested from full‐term delivery UCs. The isolated MSCs were cultured with Mesenchymal Expansion Medium (R&D Systems Inc., Minneapolis, MN, USA) in a 5% CO₂ incubator at 37°C. For flow cytometric analysis, the cells were incubated with the following antibodies: anti‐human CD105‐PE (eBioscience Inc., CA, USA), anti‐human CD34‐FITC (eBioscience Inc. CA, USA), anti‐human CD31‐FITC (BD Pharmingen Inc., San Diego, CA, USA). Then, the cells were washed with PBS and were analysed with a Calibur flow cytometer (BD Pharmingen Inc.).

Single frozen vials of the SVF were thawed at 37 °C in a water bath and the content was transferred to 9 mL of RPMI 1640 GlutaMAX medium supplemented with 10% FBS and 1% AA (all from Gibco) before centrifugation at 300 g for 5 min. Cells were incubated first with human Fc receptor blocking solution (1:10, Biolegend, 422302) and efluor 780 fixable viability dye (1:1000, eBioscience, San Diego, CA, USA, 65-0865-14,) in PBS for 5 min at 4 °C. Afterwards, the antibodies were directly added on the cell suspension and incubated for 15 min at 4 °C. For staining of LECs, the following antibodies were used: BV421 anti-human CD45 (1:200, Biolegend, 304032), FITC anti-human CD31 (1:20, BD Pharmingen, NJ, USA, 555445,), Pe-Cyanine 7 anti-human podoplanin (1:200, Biolegend, 337014) and PE anti-human Nectin-2 (1:200, Biolegend, 337409) or the isotype PE anti-mouse IgG1 (1:200, Biolegend, 400114). For staining of DCs, the following antibodies were used: BV421 anti-human CD45 (1:200, Biolegend, 304032), APC anti-human CD86 (1:200, Biolegend, 305411), FITC anti-human HLA-DR (1:200, Biolegend, 307603) and PE anti-human Nectin-2 (1:200, Biolegend, 337409) or the isotype PE anti-mouse IgG1 (1:200, Biolegend, 400114). Cell suspensions were washed once prior to recording on a CytoFlex S instrument (Beckman Coulter). Data were analyzed using FlowJo software version 10.8.1 (BD Life Sciences).

The surface MSC phenotype was analyzed using flow cytometry analysis. 3D Petri dishes were inverted and centrifuged at 300 g for 10 min to remove spheroids from the agar. Spheroids and 2D MSCs were then Trypsinized using 2.5% Trypsin (Gibco) for 15 min, and 50,000 cells/tube were incubated with specific primary antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): FITC-anti-human CD31, FITC-anti-human CD34 (BD Bioscience, San Diego, CA, United States), PE-anti-human CD90, and PE-anti-human CD105 (BD Pharmingen, San Diego, CA, United States). Cells were also stained with the correlate IgG isotype controls conjugated with FITC (BD Bioscience) or PE (BD Pharmingen). Stainings were performed for 30 min at room temperature (RT) in the dark. Samples were run on a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, United States), and data were analyzed using FlowJo software (BD Biosciences).