Anti cd90 pe

Total bone marrow cells were flushed from the femurs and tibias of rats (2- to 4-month-old) with culture medium; the rats were sacrificed via sevoflurane overdose as previously described [31 (link)]. Complete L-DMEM containing 15% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin was used to resuspend the BMSCs, and then, the BMSCs were incubated in a humid chamber. At 48 h, the first medium change was performed to remove the nonadherent cells. When the cells reached 90% confluence, they were harvested with 0.25% trypsin (Sigma) and passaged at a ratio of 1 : 2. FCM was performed to assess the BMSC surface markers. The cells were incubated with the following fluorochrome-conjugated primary antibodies (all from BioLegend, USA): anti-CD90-PE, anti-CD29-APC, and anti-CD45-PE. BMSCs from passage (P) 3 to P5 were used for subsequent experiments.
The cells were stimulated with hypoxia, and cell viability was detected [32 (link)]. Approximately 5 × 10⁵ BMSCs suspended in L-DMEM were plated in 150 mm-diameter culture dishes. The cells were then separately cultured under the conditions below for 12, 24, 48, 72, 96, or 120 h: 10% exosome-free FBS with hypoxia (94% N₂, 5% CO₂, and 1% O₂ gas mixture). BMSC viability was analyzed with CCK-8 assays. Briefly, adherent cells were digested with 0.05% trypsin and collected for CCK-8 assay according to the manufacturer's instructions.

RA FLS were grown to 90% confluence on 8-well glass chamber slides. Cells were fixed with 1% Formalin and blocked with Fc block (10% human serum/10% mouse serum in PBS). Cells were incubated for 1hour at room temperature with anti-CD13-FITC (1D7) 1μg/100μl and anti-MMP14-PE (clone128527, R&D, Minneapolis, MN) 1.67μg/100μl or anti-CD90-PE 1μg/100μl (3E10, Biolegend, San Diego, CA). All experiments also included staining with MsIgG isotype controls (MsIg-FITC [eBiosciences, San Diego, CA], MsIg-PE [Biolegend, San Diego, CA]) at the same concentrations. The nuclei were counter stained with DAPI at 1μg/ml. Cells were mounted using Pro-gold anti-fade media (Life Technologies, Carlsbad, CA). Images were taken with an Olympus Fluo-View 500 confocal microscope system (University of Michigan Microscopy and Image Analysis Core) at 600x and1000x. All images were corrected for background, thresholds were determined by DAPI alone, MsIg-FITC alone, and MsIg-PE alone. Co-localization was analyzed with ImageJ using the plug-in “Colocalization”, by Pierre Bourdoncle, Institut Jacques Monod, Service Imagerie, Paris.