Flow cytometry instrument

Four young inbred male BALB/c mice were euthanized using ketamine (100 mg/Kg; Alfasan, Netherlands) and xylazine (10 mg/Kg; Alfasan, Netherlands) to obtain ASCs. The epididymal fats were then isolated and washed using sterile PBS containing penicillin-streptomycin (Sigma-Aldrich, USA) antibiotics. After mesh-based slicing of fat tissues (1 mm pieces), they were placed into a Falcon tube containing 9 mL DMEM medium (Gibco, USA), and 1 mL collagenase (Gibco, USA), and were shaken for 30-40 min. The resultant suspension was centrifuged at 1,200 rpm for 10 min, and the cellular pellet was cultured in 5 mL DMEM supplemented with 15% FCS and placed in a standard cell culture incubator. The culture medium was replaced with fresh DMEM containing 10% serum and antibiotics every three days. Osteogenic and adipogenic potentials, at passage 3, were evaluated using Alizarin-Red (Sigma-Aldrich, USA) and Oil-red O (Sigma-Aldrich, USA) staining, respectively.
For detecting specific surface markers, ASCs (1,000,000 Cells), at passage 3, were exposed to fluorescein isothiocyanate (FITC)-conjugated antibody CD90 (ab11155), CD44 (ab30405), CD73 (ab239246), and CD34 (ab18227) diluted in PBS for 40 min on ice. After washing with PBS, the fluorescence intensity of ASCs was measured using a flow cytometry instrument (Becton Dickinson, USA), and the data were analyzed using Flow-Jo software.

3T3 preadipocytes were cultured in 6-well plates at a density of 6×10⁵ cells per well and transfected with miR-129-5p mimic or NC 24 h later by using Roche Transfection Reagent. 3T3-L1 preadipocytes were washed three times with PBS and harvested by trypsin digestion 48 h after transfection. Harvested preadipocytes were fixed in 70% cold alcohol overnight at −20°C and treated with 1 mg/mL RNase A at 37°C for 45 min, then 50 mg/mL propidium iodide (PI) was used for staining for 30 min. Finally, the cell cycle of these cells was analyzed by a flow-cytometry instrument (Becton Dickinson, Franklin Lakes, NJ).

Porcine intramuscular preadipocytes being the stable phase of cell growth, cells were seeded in 6-well culture plates at a density of 1.6 × 10⁶ cells per well. After 24 h, cells were transfected with miR-425-5p agomir/NC by X-tremeGENE HP siRNA Transfection Reagent (Roche), and cells were washed three times with PBS, harvested at 48 h post-transfection, and then stained with DNA staining solution (CCS01; Multi Sciences, Hangzhou, China), and left to incubate for 30 min. Finally, a flow cytometry instrument (Becton Dickinson, Franklin Lakes, NJ, USA) was used to analyze the proliferation phase of the cells.

For apoptosis detection, transfected cells were staining with 7AAD and Annexin V-PE (Bio-Box, Nanjing, China). For cell cycle detection, transfected cells were fixed in 70% ethanol overnight at 4°C and then washed with phosphate buffered saline (PBS). After washing, cells were resuspended in 500 μL PBS with PI (50 mg/L) and RNaseA (100 mg/L) (Sigma, St. Louis, MO, USA) and analyzed with a Becton Dickinson (BD) flow cytometry instrument (BD, New Jersey, USA). FlowJo software (FlowJo, Ashland, OR, USA) was used for cell cycle and apoptosis analysis.

The apoptosis rate of PC12 cells with corresponding treatment in each group was detected using Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (C1062L; Beyotime). Following the manufacturer’s guideline, adherent PC12 cells were digested using trypsin (C0202; Beyotime) and rinsed off. Then, cells were collected, centrifuged, and re-suspended using phosphate-buffered saline (PBS). Cell number was counted. About 5 × 10⁴ re-suspended PC12 cells were centrifuged. The precipitate was re-suspended again using 195 μL of Annexin V-FITC binding buffer. Then, the re-suspended cells were added with 5 μL of Annexin V-FITC and 10 μL of PI and incubated in the dark at RT for 15 min. Cell apoptosis was detected by flow cytometry instrument (BD Biosciences), and the data were analyzed with FlowJo software (Stanford University, San Francisco, CA, USA).

For the cell cycle analysis, the stable EC cells were synchronized in FBS-free medium for 12 h and then recovered in fresh medium for 12 and 18 h, respectively. Afterwards, these cells were fixed with 95% ethanol at 4 °C overnight. The next day, the pellets were washed with cold PBS for three times and then stained with PI/RNase buffer (BD Pharmingen^TM) for 10 min at room temperature, followed by the analysis on the flow cytometry instrument (BD Bioscience). For the apoptosis analysis, the cells were stained with Annexin-V and PI (Invitrogen) in turn at room temperature, followed by the flow cytometry analysis according to our previous performance^{29 (link)}.

After digesting and washing, the fourth-generation (p4) PDLSCs were used to detect specific surface markers under flow cytometry instrument (BD Biosciences, NJ, USA). Mesenchymal stem cell surface antibodies (CD90, CD44, and CD105) (BD Biosciences, NJ, USA) and hematopoietic stem cell surface antibodies (CD45, CD31) (BD Biosciences, NJ, USA) were selected to examine.