1. Remove 13.5 day pups and transfer into PBS- solution.
2. Remove limbs, internal organs etc.
3. Wash in another PBS- solution.
4. Mince using fine forcep and blade for 5 minutes.
5. Transfer into trypsin-EDTA solution. Further mince for a few minutes.
6. Transfer the solution into a beaker and stir with magnetic stirrer for 20 minutes.
7. Filter into conical tube using nylon (as substitute for cell strainer)
8. Add DMEM with 3% penicilin-streptomycin and 10% FBS at ratio of 1:1.15 DMEM
9. Centrifuge for 5 minutes.
10. Prepare culture dish by adding DMEM solution
11. Aspirate the supernatant out from the conical tube. Add DMEM into the pellet and suck in and out to produce single cells.
12. Transfer the cells to the culture dishes. Incubate.
13. The medium is replaced the next day.
1. Remove media from MEF culture dishes.
2. Wash 2-3 times with PBS-.
3. Deatch the cells using trypsin for 3-5 minutes.
4. Add 1ml of DMEM with 1% penicilin-streptomycin and 10% FBS into conical tube.
5. Fill the conical tubes with detached cells. Add with DMEM.
6. Centrifuge for 5 minutes.
7. Prepare culture dishes by adding DMEM.
8. Aspirate and discard supernatang from conical tubes.
9. Add 2 ml DMEM into tube and suck in and out to dissociate the cells.
10. Place the cells into culture dishes.
11 Incubate and replace the medium 2 days later.
i'm trully grateful of any advices. thank you.
Edited by JWChung, 02 September 2010 - 07:30 AM.