Cd 90 apc cy7

Immunostaining and flow cytometry analyzes (FACS) were performed to detect and confirm the presence of specific cell surface antigens characteristic for hASCs. All mouse antibodies used [CD 29-PE (BD 562801), CD 34-PE-Cy7 (BD 560710), CD 44-APC (BD 559942), CD 45-PerCP (BD 557235), CD 73b-FITC (BD), CD 90-APC-Cy7 (BD 561401), CD 105- Percp-Cy5.5 (BD 560819) and streptavidin (BD 554066)] were purchased from BD Biosciences (USA). Fluorochrome-conjugated mouse immunoglobulin was used as isotype control. Single cell suspensions of hASC were subsequently analyzed on a Becton–Dickinson FACSCalibur flow cytometer to obtain at least ten thousand cells. Samples were analyzed by FlowJo software (TreeStar, USA).

On the 2nd passage, cells were analyzed by flow cytometry with BD FACSAria cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA) using anti-mouse monoclonal antibodies: CD90 APC-Cy7 (BD Biosciences, cat. no. 561401, Franklin Lakes, NJ, USA), CD105 APC (Invitrogen, cat. no. 17-1051-82, Carlsbad, CA, USA), CD73 PE (BD Biosciences, cat. no. 550741, Franklin Lakes, NJ, USA), CD44 PE (BD Biosciences, cat. no. 553134, Franklin Lakes, NJ, USA), CD45 PE (Thermo Fisher Scientific, cat. no. MA1-10233, Waltham, MA, USA), CD34 Alexa Fluor^® 647 (BD Biosciences, cat. no. 560230, Franklin Lakes, NJ, USA).

Flow cytometry (FACS AriaII, BD, USA) was used to confirm the identity of ADSCs by detecting the expressions of cell surface markers, including CD29-FITC (102205, Biolegend), CD90-APC-Cy7 (561641, BD), CD105-BB515 (564744, BD), CD44-PE-Cy5 (103009, Biolegend), CD31-APC (551262, BD), CD34-FITC(11-0341-82, eBioscience), and CD45-APC (559864, BD) [25 (link)]. Third-passage ADSCs were digested with trypsin (Sigma-Aldrich, United States), re-suspended in PBS, and counted using a hemocytometer. A total of 1×10⁶ cells were treated with the fluorochrome-conjugated antibody at a dilution ratio of 1:10 for 30 min at room temperature with protection from light. S1 Table shows the antibodies used in these experiments.