Passaging cells Pour out media from flasks. Wash with Hanks. 5 ml per flask. Tilt around then dump. Add 4 ml of Trypsin / EDTA to each flask. Tip, then bang. Add 20% FBS NCTC media and tilt. 4 ml per flask. Scrape (most of the small cells are already released; proliferative cells lift up easy, differentiative cells are more adherent). Place contents into a 50 ml Falcon tube. Spin at 1000 RPM for 5 min (#5 setting = 1000) Dump off supernatant. Add enough media to allow 2 ml of cells per flask (RCHO-cells start to differentiate at day two. At that time a flask contains ca. 1 million cells. They are spread by 1 : 3). Each flask should have 8 (or 10) ml of 20% FBS NCTC media + 2 ml of cells for a total of 10 (or 12) ml. |
Freezer and Cell Line Database Very useful Microsoft Access database program (486kb) for managing your cell lines stored in freezer. It's easy to use and has a very nice screen. 20040724154009292.rar |
Defrosting Eukaryotic Cells Frozen In Liquid N2 Reagents / Solutions
Protocol 1. Make 20ml growth media/10% FCS for each vial of cells to be thawed. 2. Place a small sandwich box containing warm (~ 37℃) water in the CO2 incubator. 3. Remove vial(s) of cells and thaw rapidly by placing in an expanded polystyrene 'floater' in the water bath. Transfer cells to a universal. 4. Add 10ml media/10% FCS very slowly. First ml should take ~ 1 min, second ml should take ~ 45 seconds etc. 5. When cells are resuspended in 10ml spin down in bench-top centrifuge and resuspend in 10 ml media/10% FCS (this does not need to be added slowly). 6. Place cells in large flask and allow to settle for ≥ 2 hours. 7. Take a sample, assess % viability and cell number. 8. Allow to grow overnight. 9. Assess cell number and viability, if the cell density is too high for the normal grwoth of the cell line add further normal growth media (e.g. RPMI1640/10% FCS. |
Cell Freezing Reagents / Solutions
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