Reagents and materials:
1. Differentiation medium: DMEM/F12 (1:1), 1% ITS (insulin, transferrin, selenium; V/V), TGF-β1 1ng/ml, HEPES 10mmol/L;
2. Trypsin/EDTA: trypsin (0.05%) and EDTA (0.53mmol/L) PBSA;
3. PBSA: Dulbecco's phosphate buffer without Ca2+, Mg2+;
4. Centrifuge tube, 15ml;
5. Ordinary utensils, 30 ml, or a conical bottom centrifuge tube (50 ml);
experimental method:
1. Aspirate the growth medium from the culture flask and discard it;
2. Wash the PBSA after washing;
3. Add sufficient trypsin/EDTA to cover the bottom of the flask;
4. Incubate at 37 ° C for 5-10 min (observe under the microscope whether the cells are shed);
5. Resuspend the cells, place them in a 15 ml centrifuge tube, and centrifuge at 300 g for 5 min;
6. Wash with 1 ml of growth medium and transfer to a common vessel or centrifuge tube;
7. Count the cells using a blood cell counting plate;
8. Repeat centrifugation (620 g, 10 min) and discard the supernatant;
9. Dilute it to 1 x 106 cells/ml with differentiation medium;
10. Dispense into a new Eppendorf tube at 1 ml/tube;
11. Centrifuge at 300g for 5min, the supernatant remains intact;
12. Place in a 37 ° C incubator;
13. Change the medium every 2-3 days;
14. Culture 2-3 weeks of chondrogenesis;
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