Pe anti mouse cd29

For flow-cytometric analysis, cells were trypsinised, resuspended in FACS buffer (1% FBS in PBS+1 mM CaCl₂+1 mM MgCl₂) and labelled with one of the following antibodies (all used at 1:200 and, unless stated otherwise, purchased from eBioscience, Hatfield, UK): PE-anti-mouse Flk1, PE-anti-mouse CD49a (Cambridge Bioscience, Cambridge, UK); PE-anti-mouse CD49b; PE-anti-mouse CD49e; PE-anti-mouse CD51; PE-anti-mouse CD29; PE-anti-mouse CD61; PE-anti-mouse integrin beta 5; PE-anti-mouse CD31; FITC-anti-mouse ICAM2; PE-anti-mouse VECAD; appropriate PE/FITC-labelled isotype-matched controls were from eBioscience. In the case of Flk1 analysis, cells were stimulated with 30 ng/ml VEGF at 37°C over a 60-min time course before trypsinisation.

Cells were obtained from the tibia and femur bone marrow aspirates from WT and MMTV-PyMT mice, and went through a 70 μm cell strainer, and resuspended in PBS supplemented with 2% FBS. Then cells were suspended in 50 μL staining buffer (PBS containing 2% FBS), with monoclonal antibodies and incubated for 20 minutes at 4°C. Finally, cells were washed twice and resuspended in 200 μL of PBS, and then analyzed on a flow cytometer (Cytoflex, Beckman Coulter). The antibodies used for Flow cytometry as follows: Annexin V-FITC (BD Biosciences, 556547), 7AAD (eBioscience 00-6993-50), PE anti-mouse CD45 (BioLegend 147712), FITC anti-mouse CD11b (eBioscience 11-0112-85), PE anti-mouse Ly6G (eBioscience,12-5931-83), eFluor 450 anti-mouse Lineage (eBioscience 88-7772-72), PE-Cy7 anti-mouse Sca1 (BioLegend 108114), PE anti-mouse CD29 (eBioscience 12-0291-81), FITC anti-CD44 (eBioscience, 11-0441-85), PE anti-mouse CD44 (eBioscience 12-0441-83), PE anti-mouse CD73 (Biolegend 117203).

Characterization of the surface markers of cells was performed by using flow cytometry. MSCs digested in a dish with 0.25% Trypsin (Biosharp) were suspended in staining buffer (PBS, 1% FBS) and 1 × 10⁵ cells/sample were incubated with fluorescently labeled antibodies (all 1:200) for 30 min at 4 °C. The antibodies used for flow cytometry were as follows: PE anti-mouse CD29 (eBioscience), PE anti-mouse Sca-1 (Biolegend), PE anti-mouse CD140a (eBioscience), PE anti-mouse CD44 (eBioscience), PE anti-mouse CD73 (eBioscience), PE anti-mouse MHC II (eBioscience), PE anti-mouse CD45 (Biolegend), PE anti-mouse CD31 (eBioscience), PE anti-mouse CD34 (Biolegend), PE anti-mouse CD11B (eBioscience), PE anti-mouse ICAM-1 (Biolegend), and BV421 anti-mouse VCAM-1 (BD Pharmingen). For intracellular staining, cells stained with cell surface antibodies (PE anti-mouse CD3 (Biolegend)) were fixed, permeabilized using foxp3/transcription factor concentrate and diluent kit set prior to incubation with antibodies directed at intracellular antigens (APC anti-mouse KI-67 (Biolegend)). Fluorescence intensity was measured by flow cytometry (Cytoflex, Beckman Coulter). Data were analyzed with FlowJo v10 software and Cytexpert software.