Anti cd34

To prepare the cells for immunofluorescence staining, hAMSCs were washed with 1x DPBS (Sigma, D5773) and fixed with 4% paraformaldehyde (EMPROVE®, Germany, 1,040,051,000) for 5 min at 4 °C. The cells were then washed again with 1x DPBS and permeabilized with 0.1% Triton-X100 in 1x DPBS for 5 min at 4 °C. The following primary antibodies were used for staining: anti-CD44 (1:50), anti-CD73 (1:200), anti-CD90 (1:100), anti-CD105 (1:100), anti-CD34 (1:250), anti-CD11b (1:250), anti-CD19 (1:250), anti-CD45 (1:250), and anti-HLA-DR (1:250) (Invitrogen™). Differentiating cells were stained with Sarcomeric alpha actinin monoclonal antibody (EA-53) (Invitrogen, MAI-22863, 1:500) and Anti-Cardiac Troponin T (abcam, ab8295, 1:1000) primary antibodies, Goat Anti-Mouse IgG H&L secondary antibody (abcam, ab150113, 1:1000), and DAPI (Biotium, 40043, 1:1000) to visualize nuclei. The antibodies were diluted in PBS containing 1% bovine serum albumin (Sigma, Germany, A2153) and cells were incubated with the primary antibodies overnight at 4 °C. The cells were then washed with PBS. To observe the results of immunofluorescence staining in cardiac genes, we used Leica DM2500 Optical Microscope (Leica Microsystems, Germany) equipped with a Canon EOS 7D camera (Canon, Japan). As part of the cardiac gene expression analysis, human cardiomyocytes (Promocell, C-12810) were used as positive control.

Single cells were collected with 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then resuspended in cold PBS at a concentration of 5×10⁶ cells/mL. Then, the cells were incubated in the dark on ice for 30 min using antibodies including anti-CD29, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105 and anti-CD146. All antibodies were purchased from BioLegend, Inc. Subsequently, cells were twice washed and 500 μL PBS resuspension for each sample. The study was performed by Becton-Dickinson Accuri C6 (BD Biosciences, San Jose, CA, USA). FlowJo (version 10.0.7 r2) was used for the data analysis.

The following commercial antibodies were used: mouse hematopoietic lineage eFlour 450 cocktail, PerCP-Cy5.5-anti-CD45.1, FITC-anti-CD45.2, Alexa Fluor 700-anti-IL-7Rα, FITC-anti-Ly6A/E (Sca-1), PE-anti-CD117 (c-Kit), APC-eFluor780-anti-CD48, anti-CD3, anti-CD19, FITC-anti-CD11b, PE-anti-Gr1, FITC-anti-B220, Pecy5-anti-CD3ε, APC-anti-Flt3/CD135, anti-F4/80, anti-Gr-1, and anti-CD34 were purchased from eBioscience. PE-cy7-anti-CD150 was obtained from BioLegend. PerCP-Cy5.5–conjugated goat anti–rat IgG and APC-Cy7–conjugated goat anti–rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. Anti–β-actin was from Sigma-Aldrich. Antibodies against Insr-β, phosphorylated Insr-β (Tyr1150/1151), insulin, mTOR, Stat3, S727 phosphorylated Stat3, Y705 phosphorylated Stat3, S6K, and phosphorylated S6K were purchased from Cell Signaling Technology. Donkey anti–rabbit or anti–mouse secondary antibodies conjugated with Alexa Fluor 488, 594, or 405 were purchased from Molecular Probes. HRP-conjugated secondary antibody was obtained from Santa Cruz Biotechnology, Inc. Propidium iodide (PI), Annexin-V, insulin, rapamycin, and streptozotocin (STZ) were purchased from Sigma-Aldrich.