Cell Transformation Group:Protocols/Cell Culture: Difference between revisions
No edit summary |
No edit summary |
||
Line 7: | Line 7: | ||
:1. Lift cells when 70 – 80% confluent and resuspend in 10% FCS/DMEM (~8 ml) (75 cm<sup>2</sup> FLASK = 1 X 10<sup>7</sup> Cells/ml).<BR> | :1. Lift cells when 70 – 80% confluent and resuspend in 10% FCS/DMEM (~8 ml) (75 cm<sup>2</sup> FLASK = 1 X 10<sup>7</sup> Cells/ml).<BR> | ||
:2. Spin at 250 g for 5 min. Discard S/N. Resuspend in 2 ml of DMEM (or 4 ml for large flasks). Put on ice.<BR> | :2. Spin at 250 g for 5 min. Discard S/N. Resuspend in 2 ml of DMEM (or 4 ml for large flasks). Put on ice.<BR> | ||
:3. Prepare freezing mix: per ml add 600 μL DMEM (+10%FCS | :3. Prepare freezing mix: per ml add 600 μL DMEM (+10%FCS) with 200 μL FCS and 200 μL DMSO (add DMSO [flammable goods cupboard] last as a precipitation forms) and chill on ice. This is the freezing mix. IMPORTANT - adjust the final volume of freezing mix (need 1 mL per mL of resuspended cells).<BR> | ||
:4. | :4. Mix at a ratio of 1:1 the freezing mix and cell mix, by adding dropwise the freezing mix to the cells. gently shaking to ensure mixing.<BR> | ||
:5. 1 ml of mix to each cryovial on ice. transfer to “Mr Frosty” and freeze at -70 °C for 24 hrs. Make sure to mark off Mr Frosty usage by crossing out number on lid. Refill with isopropanol after 5 usages.<BR> | :5. Add 1 ml of mix to each cryovial on ice. transfer to “Mr Frosty” and freeze at -70 °C for 24 hrs. Make sure to mark off Mr Frosty usage by crossing out number on lid. Refill with isopropanol after 5 usages.<BR> | ||
:6. | :6. Transfer to liquid nitrogen and add to cell lines log book.<BR> | ||
===TO THAW:=== | ===TO THAW:=== |
Revision as of 20:59, 18 June 2009
<owwmenu> image="A549_web.jpg" font="arial, helvetica, sans-serif" bold="1" color="blue" bgcolor="Cornsilk" hovercolor="lightcyan"
bghovercolor="rosybrown" topFontSize="15" fontSize="10" pagewidth="800" lab="Cell Transformation Group">
Home=#, Home=http://openwetware.org/wiki/Cell_Transformation_Group
Members
Research=#,Ongoing=Ongoing_Research
Protocols=#,Cell_Culture=Protocols/Cell Culture,Protein=Protocols/Protein,RNA_and_DNA=Protocols/RNA and DNA
Resources=#,Cell Lines=Resources/Cell Lines,Virus_Stocks=Resources/Virus Stocks,Glycerols=Resources/Glycerols,Antibodies=Resources/Antibodies
Links=#, Sponsors=Links/Sponsors
</owwmenu>
FREEZING AND THAWING CELLS
TO FREEZE:
- 1. Lift cells when 70 – 80% confluent and resuspend in 10% FCS/DMEM (~8 ml) (75 cm2 FLASK = 1 X 107 Cells/ml).
- 2. Spin at 250 g for 5 min. Discard S/N. Resuspend in 2 ml of DMEM (or 4 ml for large flasks). Put on ice.
- 3. Prepare freezing mix: per ml add 600 μL DMEM (+10%FCS) with 200 μL FCS and 200 μL DMSO (add DMSO [flammable goods cupboard] last as a precipitation forms) and chill on ice. This is the freezing mix. IMPORTANT - adjust the final volume of freezing mix (need 1 mL per mL of resuspended cells).
- 4. Mix at a ratio of 1:1 the freezing mix and cell mix, by adding dropwise the freezing mix to the cells. gently shaking to ensure mixing.
- 5. Add 1 ml of mix to each cryovial on ice. transfer to “Mr Frosty” and freeze at -70 °C for 24 hrs. Make sure to mark off Mr Frosty usage by crossing out number on lid. Refill with isopropanol after 5 usages.
- 6. Transfer to liquid nitrogen and add to cell lines log book.
TO THAW:
Speed is essential for the first few steps as DMSO is harmful to cells and the less time they are in contact with it the better.
- 1. Remove cells from liquid nitrogen. Thaw quickly at 37 °C.
- 2. Pipette into warm complete DMEM (10 ml).
- 3. Spin at 250 g for 10 mins.
- 4. Resuspend cell pellet in 5ml of DMEM and transfer to medium flask (75 cm2) containing 20 ml of DMEM.
- 5. Incubate in 37 °C CO2 incubator. Delete from cell lines log book.
If Thawing Lots of Samples (>5)
- 1. Fill chilly bin with dry ice from -70 °C freezer
- 2. Remove cells from liq N2 and place on dry ice
- 3. Place chilly bin (with cells) in -70 °C
- 4. Pre-label a medium flask† and a plastic universal for each cell line being brought up. Add appropriate amounts of DMEM to each
- 5. Take 4 (or appropriate number) vials out at a time and thaw at 37 °C
- 6. Pipette entire contents of thawed vial into its corresponding universal
- 7. Spin at 250 g for 10 min [removes DMSO]
- 8. Use a 10ml pipette to resuspend the cell pellet in 5 ml DMEM and transfer suspension to a medium flask
- 9. Incubate at 37 °C. Delete from cell lines log book.