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Message | User | Date(yyyy-mm-dd) |
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lung cancer cells.. | kath | 2007-11-04 | Click here to register. |
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| hi...i had problem about lung cancer cells, recently i received new lung cancer cells and had culture it adhere..but the problem is now, when i put 1ml tripsin to detach the cell and incubate it for 5 min, the cell did not detach. what happen here? help.... | | |
| r.anggelia | 2007-11-05 | Click here to register. |
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 | i think the cell were cultured too long so it was attach the surface of flask hardly.
perhaps this procedure could help you
1. the cell need more time / much trypsin to incubate
2. trypsin were incubate on 37 OC before we used.
3. after incubate cells spray the cells with trypsin using pippette
4. you need to shake hardly the flask with your hand. (Sometimes this step could help cells to detach)
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| Diane | 2007-11-08 | Click here to register. |
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| If you haven't already tried this:
1. Wash plate with PBS same volume that you culture with before adding trypsin.
2. Make sure trypsin is prewarmed and is new lot. Make sure you are storing your trypsin correctly.
3. Add 2 mL trypsin and place plate in incubator. After 3 minutes, start shaking plate as you watch under scope. You should see them start to round up and float.
4. Stop Trypsinization by adding to 10x Media with serum. Collect culture in a tube
5. Count and centrifuge, reseed. Good luck |
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