Anti cd90 antibody

P3 ASCs were resuspended following digestion with 0.25% trypsin and washed three times with phosphate-buffered saline (PBS). A minimum of 1 × 10⁵ cells were collected from each sample. Samples were incubated with Anti-CD34 antibody (NBP2-29455; Novus), Anti-CD45RA antibody (561886; BD Pharmingen), Anti-CD73 antibody (orb629433; BIORBYT), Anti-CD90 antibody (561973; BD Pharmingen), and Anti-CD105 antibody (NB500-452; Novus) in the dark at room temperature for 30 min. The samples were subjected to flow cytometry, and data were analyzed using Paint-A-Gate Pro™ software (FACSCalibur™; BD Biosciences).

Transplanted scaffolds were harvested from defect area after EMC and IMC post-implantation for 1 week. The scaffolds were minced and cell were flushed with Ca²⁺ and Mg²⁺ free PBS with 2% fetal bovine serum for at least 3 times and then incubated with Anti-CD90 antibody, a surface marker of MSCs, at 1:100 dilution for 30 mins (BD Biosciences). For cell culture, the cells were trypsinized after cultured for 3 days with DMSO or GW4869, and incubated with M2 macrophages antibodies Anti-CD68 (BD Biosciences) and Anti-CD163 (BD Biosciences) at 1:100 dilution for 30 mins. DAPI was used to exclude dead cells. The flow cytometry analysis of positive cells was performed on the Accuri-C6 (BD Bioscience).

Rat BM-MSCs were detached, collected, and centrifuged at 300 × g for 5 min and then suspended in the expansion medium. The viability of BM-MSCS was evaluated in an aliquot using Trypan blue stain by counting the stained live cells and excluding the unstained dead cells as reported by Al-Mutairi et al. [25 ]. For BM-MSC, surface antigen phenotypic analysis and cell markers were assessed [26 (link)]. After being washed twice with phosphate-buffered saline (PBS, PH 7.4; 137-mM NaCl, 2.7-mM KCl, 10-mM Na₂HPO₄, and 1.8-mM KH₂PO₄) (Lonza, Germany) containing 1% bovine serum albumin (ALB) (Sigma-Aldrich), 0.2×10⁶ cells were stained with anti-CD34, anti-CD45, anti-CD29, anti-CD73, and anti-CD90 antibodies (BD Biosciences, USA). A FACSCalibur flow cytometer (BD Biosciences) was used to examine conjugated cells as well as determine the proportions of positive and negative populations. A negative sample with untreated isotype cells was used as a control.