Pe anti rat cd45

TDSCs were incubated with fluorescent primary antibodies on ice for 60 min, washed three times with phosphate-buffered saline (PBS), and analyzed by flow cytometry. The following fluorescent primary antibodies were used: FITC anti-rat CD29, FITC anti-rat CD44, PE anti-rat CD45, and PE anti-rat CD90 (BioLegend, San Diego, CA, United States). FITC isotype control and PE iso control were added to the NC samples (BioLegend, San Diego, CA, United States).

The phenotypic properties of BMSCs were determined on the basis of the expression of CD90, CD29 and the absence of the pan-hematopoietic marker CD45 at passage 1 and 3. Briefly, BMSCs (0.2 × 10⁶ cells) were incubated with Phycoerythrin (PE)-conjugated antibodies or isotype-matched control immunoglobulin Gs (IgGs, 1 μg each) diluted in PBS containing 2% FCS, 2 mM EDTA, and 0.01% sodium azide (PFEA buffer) at 4°C for 45 minutes (BD Biosciences). For flow cytometry, the following antibodies were employed according to the supplier's recommendations: PE anti-mouse/rat CD29 (Clone: HMβ1-1, BioLegend); PE anti-rat CD45 (Clone: OX-1, BioLegend), and PE anti-rat CD90 (Clone: OX-7, BioLegend, all from San Diego, CA) and their isotype controls: PE Armenian Hamster IgG (CD29) and PE Mouse IgG1 (CD45, CD90) from Biolegend.
Samples were analyzed using a flow cytometer FACS Calibur (BD Biosciences) operated by CellQuest software and at least 20,000 events were collected per sample. Data were analyzed using WinMDI software (Version 2.8). Forward and side scatter profiles were obtained from the same samples.

The third passage cells were collected to identify the phenotypes. When rBMMSCs washed with PBS twice and filtered to resuspend in 100µl PBS, four kinds of primary antibodies including PE anti-rat CD44 (Abcam, ab23396, USA), PE anti-rat CD29 (Biosciences, 562154, USA), PE anti-rat CD45 (Biolegend, 202207, USA) and FITC anti-rat CD34 (SantaCruz, sc-7324, USA) were added in and incubated at 4°C for 30min without light. The antigen expressions can be analyzed by flow cytometer (BD FACSCalibur, USA) according to the manufacturers’ instructions.