OCCM30 Sub-Culture and counting protocol

1. Prepare 5% FBS DMEM medium

2. Prepare 4 tube (Trypsin, DMEM, Cells, DPBS)
3. Prepare 4 6cm Culture Dish
4. Prepare hemocytometer
5. Remove medium from old culture dish
6. Put 2 ml DPBS Immidiately
7. Aspirate DPBS
8. Put 0,3 ml Trypsin
9. Check on the microscope (to confirm the cells)
10. Put 1,7 ml 10% FBS DMEM into the culture dish
11. Centrifuge the cell suspension for 5min @1500rpm, 4C temperature.
12. Discard the supernatant and gently flick the cell pellet (to break up pellet), add 3 ml 5%
FBS DMEM medium
13. Inject 10l Cell Suspension to hemocytometer
14. Check and count the cells on the microscope
15. The cell number is ___ x 104 Cells/ml
16. Add ___ ml 5% FBS DMEM Medium into the cells tube until the amount of cell
number is 14 x 104 Cells/ml
17. Prepare 3 ml 5% FBS DMEM into each culture dish
18. Transferr 1 ml Cells suspension so it has 14 x 104 Cells/dish
19. Check on the microscope (to confirm the cells)

Materials needed:
1. 10% FBS DMEM Medium 1,7ml
2. 5% FBS DMEM Medium __ ml
3. Hemocytometer
4. Trypsin 0,3 ml
5. DPBS 2 ml
6. 4 pcs 6cm culture dish
7. 4 pcs 15ml Tube