Anti cd90 fitc

At confluence, the cells derived from the Rigenera digestion method or the lipoaspirates from the digestive solution were detached with trypsin-EDTA (200 mg/L EDTA, 500 mg/L trypsin; Cambrex, Milan, Italy). At least 200,000 cells were, directly placed in incubation using fluorescent conjugated antibodies for 30 min at 4 °C then washed and re-suspended in 0.6 mL. Specimens were examined using FACS Aria II flow cytometry (Becton & Dickinson, Mountain View, CA, USA). The antibodies herein investigated were: anti-CD117 PE (c-kit) (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy); anti-CD34 FITC and PE (Miltenyi-Biotech); anti-CD90 FITC (BD Pharmingen, Buccinasco, Milano, Italy); anti-CD105 FITC (Santa Cruz, CA, USA); anti-CD73 PE (Miltenyi-Biotech); anti-CD29 PE (Miltenyi-Biotech); anti-CD31 FITC (Miltenyi-Biotech); and anti-CD45 Cy and PE (BD Pharmingen).

The hMSCs were generated from hESCs. Briefly, embryoid bodies derived from hESCs were seeded on 6-well plates coated with Matrigel (BD Biosciences, 354230), the cells were cultured for about 14 days in hMSCs differentiation medium, which was performed as previously reported (Liang et al., 2021; (link)Chu et al., 2022 (link)). The differentiation medium was changed every other day until the fibroblast-like cells appeared and reached confluent. Subsequently, the cells were then transferred into hMSCs culture medium for a continuous culture. Finally, cell sorting was conducted utilizing the fluorescence activating cell sorter (FACS) system (BD FACS Influx). CD73, CD90, and CD105 triple-positive cells were collected and further characterized by surface antigen markers, including positive marker CD44, and negative markers CD34 and CD45. The following antibodies were used for FACS: anti-CD73-PE (BD Biosciences, 550257), anti-CD90-FITC (BD Biosciences, 555595), and anti-CD105-APC (BD Biosciences, 17-1057-42), anti-CD44-FITC (BD Biosciences, 550989), anti-CD34-FITC (BD Biosciences, 555821), and anti-CD45-FITC (BD Biosciences, 555482).

Passage 3 ADSCs were digested by trypsin-EDTA and washed with PBS, then incubated with fluorescein isothiocyanate- (FITC-) conjugated and phycoerythrin- (PE-) conjugated antibodies, including anti-CD34-FITC, anti-CD44-FITC, anti-CD90-FITC, anti-CD45-PE, anti-CD73-PE, anti-CD105-PE, PE-labeled mouse IgG1 Kappa, and FITC-labeled mouse IgG1 Kappa (BD Pharmingen, USA) at 4°C for 40 min. We used flow cytometry (LSRFortessa, BD Biosciences, USA) to record and analyze the data. At least 1 × 10⁴ cells were analyzed per test.
Briefly, 12-well plates were pretreated with gelatin (Cyagen, USA) to enhance adherence. Then, to confirm the multilineage differentiation ability of ADSCs, passage 3 cells were seeded at a density of 1 × 10⁴ cells/well and cultured until 60–70% confluence was achieved. The medium of some of the wells was then changed to mesenchymal stem cell osteogenic differentiation medium (Cyagen, USA) according to the manufacturer's instructions. The medium was changed within 72 h. For adipogenic differentiation, we used mesenchymal stem cell adipogenic differentiation medium (Cyagen, USA) according to the manufacturer's instructions. Two to four weeks later, the cells were stained with alizarin red S (Solarbio, China) and oil red O (Cyagen, USA) separately to identify osteogenic and adipogenic differentiation, respectively.

Human osteosarcoma cell lines MG-63 and OS732 were purchased from the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were provided by Gibco (Grand Island, NY, USA), and recombinant human CXCL12 (SDF-1) was purchased from R&D systems (Minneapolis, MN, USA). AMD3100, a chemokine receptor antagonist for CXCR4, and Matrigel were obtained from Sigma-Aldrich (St. Louis, MO, USA). The fluorochrome-conjugated antibodies used for immunostaining - anti-CD45-APC, anti-CD29-PE, and anti-CD90-FITC - and appropriate negative controls were from BD (San Diego, CA, USA).

hMSCs were derived from hESCs as described previously (Pan et al., 2016 (link)). In brief, embryoid bodies (EBs) first formed from hESC clones in an ultralow attachment 6-well plate (Corning) in low FGF-2 hESC medium and then were transferred to a plate coated by Matrigel in hMSC differentiation medium (hMSC culture medium supplemented with additional 9 ng/mL FGF-2 and 5 ng/mL TGF-β (HumanZyme)). After 7 to 10 days, the cells became confluent and were reseeded into dishes coated by gelatin in hMSC culture medium. CD73, CD90 and CD105 tri-positive cells were sorted as hMSCs with the aid of flow cytometry. The following antibodies were used: anti-CD73-PE (BD Biosciences, 550257), anti-CD90-FITC (BD Biosciences, 555595) and anti-CD105-APC (eBioscience, 17-1057-42). The differentiation abilities of hMSCs were tested by futher differentiation into chondrocytes, adipocytes and osteoblasts (Liu et al., 2014 (link)) detected by toluidine blue (chondrocytes), oil red O (adipocytes) and von Kossa (osteoblasts) staining, respectively.

Cells were detached using trypsin EDTA (GIBCO). At least 200,000 cells were incubated with fluorescent conjugated antibodies for 30 min at 4°C, washed, and resuspended in PBS. The antibodies used in this study were anti-CD34 PE (BD Pharmingen, Buccinasco, Milano, Italy) and anti-CD90 FITC (BD Pharmingen, Buccinasco, Milano, Italy). Isotypes were used as controls. Cells were analyzed with an Accuri C6 (BD Biosciences, San Jose, CA, USA) and the data collected with FCS Express version 3 (De Novo Software). Cells were sorted using simultaneous positivity for CD90 and CD34 using a FACSAria III (BD, Franklin Lakes, NJ, USA). The purity of sorted populations was routinely 90%.