Reagents and materials:
1. 2-4 generations of mesenchymal cells;
2. HBSS/2FB;
3. Hoechst 33342 dye, 1 mg/ml water soluble;
4. Propidium iodide (PI), 2 mg/ml water soluble;
5. Verapamil, 500μg/ml water soluble;
6. DEME/2FB;
7. Trypsin/TrypLE: TrypLE Express or 0.25% trypsin, dissolved in CMF-Saline G;
8. MoFlo (or similar) high-speed cell sorter with 350nm excitation capability;
experimental method:
1. Using PriCells primary cell isolation kit for 2-4 generations of mesenchymal cells;
2. Cell count, dilute the cells to 1 × 106 / cm 2 cells / ml with DMEM / 2FB;
3. Add 5 μg/ml Hoechst 33342 dye, 37 ° C, 90 min, stir once every 20 min;
4. The control cells were incubated with 50 μg/ml verapamil for 20 min before adding Hoechst 33342 dye;
5. After staining, the cells were washed twice with HBSS/2FB and centrifuged at 4 ° C, after which the cells were resuspended on ice with cold HBSS/2FB;
6. Add 2 μg/ml PI before sorting to identify inactive cells;
7. Sort cells under sterile conditions, high speed cell sorter, and stimulate with 350 nm. Cells with reduced blue (670 nm) and red (450 nm) fluorescence were collected. The side population cells are separated from the dead cells and the cells completely labeled with the dye by the above steps and collected. The control group should ensure that the vera cell disappeared after the first incubation with verapamil;
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