Cd29 fitc

For flow-cytometry analysis of surface markers, cells were harvested during the proliferation phase at day 20 of differentiation. Cells were dissociated with 0.25% trypsin-EDTA, washed with PBS, and resuspended in flow buffer (PBS with 5% FBS). Cells were incubated with the following conjugated antibodies at 0.25 μg/10⁶ cells: immunoglobulin G1-K isotype control-fluorescein isothiocyanate (FITC) (eBioscience 11-4714-41), CD56-FITC (eBioscience 11-0566-41), or CD29-FITC (eBioscience 11-0299-41). Cells were analyzed on a Sony SH800 flow cytometer.

The culture-expanded adherent cells were analyzed by flow cytometry (FACSARIA, Becton Dickonson, USA). The antibody panel included CD29-FITC (e-bioscience, USA), CD73-PE (BD, USA), CD90-PE (BD, USA), and CD44-PE (e-bioscience, USA) as mesenchymal stromal markers, as well as their isotype controls. CD45-FITC (BD, USA), CD14-PE (BD, USA), and CD34-FITC (BD USA) were used as hematopoietic markers to exclude cells of hematopoietic origin. The relative frequencies of the cells that expressed the respective surface markers were analyzed using FACS Diva software 6.0.0 (BD) by acquiring 10,000 events for each sample.

According to the manufacturer’s instructions, CD44-PE, CD29-FITC, CD90-PE, CD11b-PE or CD45-FITC rat-specific antibodies (eBioscience, USA) was added to the bottom of tubes, after which 100 μl (6×10⁶ cells/μl) of a single-cell suspension of cultured rat BMSCs was added. The mixture was incubated for 30 min at room temperature in the dark and washed [22 ]. Positive cells were detected by flow cytometry (Becton-Dickinson, USA). Rat IgG1-FITC and IgG1-PE (eBioscience, USA) were used as isotype controls.

hADSCs were isolated from human fresh adipose liposuction liquid according to previously published methods [13 (link),16 (link)]. Flow activated cell sorting (FACS, Beckman Coulter) was carried out to identify the CD surface antigen of the isolated hADSCs at passage 3 to 5 according to published protocols [17 (link),32 (link)]. Briefly, hADSCs cultured at passage 3 to 5 were first collected by trypsin (0.25%) digestion, washed with flow wash buffer (1 × Dulbecco’s phosphate-buffered saline, 0.5% BSA, and 0.1% sodium azide), and blocked with wash buffer supplemented with mouse immunoglobulin G (25 μg/ml) for 10 minutes on ice. A total of 100 μl of this cell suspension (approximately 5 × 10⁵cells) was aliquoted per tube. Antibodies conjugated with either tricolor fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC) were purchased including CD44-APC (Cat#47-0441-80, eBioscience), CD29-FITC (Cat#11-0299-41, eBioscience), CD105-PE (Cat#12-1057-41, eBioscience), CD73-FITC (Cat#11-0739-42, eBioscience), CD34-FITC (Cat#11-0349-41, eBioscience), CD45-FITC (Cat#11-9459-41, eBioscience). Matched isotype control combinations were performed to identify the specific interaction of antigen and antibody.

Anti-CD11b-phycoerythrin (PE) cyanin 5 or fluorescein isothiocyanate (FITC), CD29-FITC, CD11b-FITC, hCD2 (hCD2)-FITC and CD18 from eBioscience, hCD163-Violet 421 and anti-mouse CD11b-violet 421, were obtained from Biolegend. mAbs against Rac1, Rac2, Cdc42 were purchased from Santa Cruz Biotechnology. Anti-FN antibody (ab2413) and anti-FN antibody [IST-9] (ab6328) were obtained from Abcam. LPS and PGN were purchased from Sigma. We purchased LTA (LTA-SA: Lipoteichoic acid from Staphylococcus aureus, Catalog# tlrl-slta), Man (LM-MS: Lipomannan from Mycobacterium smegmatis, Catalog# tlrl-lmmsl) and Zym (Zymosan: cell wall from Saccharomyces cerevisiae, Catalog# tlrl-zyn) from InvivoGen. ITGB1/Integrin β1/CD29 Antibody Blocking Peptide (β1BP) LS-E29653 (Cat No: LS-E29653; LifeSpan BioSciences, Inc). All of the TaqMan probes were purchased from Applied Biosystems. We purchased recombinant human FN protein (Cat No: ab158459) and natural mouse FN protein (Cat No: ab92784) from Abcam Biotechnology company.

The phenotype of CD105⁺CD34^- cells (1st passage) was determined using a flow cytometer (BD FACSCanto ™ BD Biosciences). The cells were incubated with appropriate antibodies directed against the following human antigens: CD29 –FITC (eBioscience), CD105 –APC, CD73 –PE, CD90 –PE-Cy7, CD44 –FITC, CD34 –PE-Cy7, CD31 –FITC, CD146 –PE, KDR–PE, LIN–FITC, CD45 –PE, HLA-DR–PE-Cy7 (BD Pharmingen), or isotype-matched control antibodies were used. The Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN, USA) was used to differentiate CD105⁺CD34^- cells into adipocytes, osteoblasts, and chondroblasts. The procedure was performed in accordance with the manufacturer’s instructions.