Smolke:Protocols/Freezer stock management and database: Difference between revisions
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(New page: Necessary Labels for All Stocks *Position on cap insert (e.g., 1A1 for Box 1, position A1) *Cell line name (e.g., CTLL-2 Parental) *Passage number (for adherent cells) *Cell number in the ...) |
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Necessary Labels for All Stocks | '''Necessary Labels for All Stocks''' | ||
*Position on cap insert (e.g., 1A1 for Box 1, position A1) | *Position on cap insert (e.g., 1A1 for Box 1, position A1) | ||
*Cell line name (e.g., CTLL-2 Parental) | *Cell line name (e.g., CTLL-2 Parental) | ||
| Line 6: | Line 6: | ||
*Initial and date | *Initial and date | ||
Lymphocyte (CTLL-2, Jurkat, and NK-92) Stocks | '''Lymphocyte (CTLL-2, Jurkat, and NK-92) Stocks''' | ||
*Count cells with Trypan Blue | *Count cells with Trypan Blue | ||
*Determine number of stocks that can be made | *Determine number of stocks that can be made | ||
| Line 17: | Line 17: | ||
*Transfer tubes to appropriate box and position in cryo container | *Transfer tubes to appropriate box and position in cryo container | ||
HEK Stocks | '''HEK Stocks''' | ||
*Trypsinize, neutralize, and count cells with Trypan Blue | *Trypsinize, neutralize, and count cells with Trypan Blue | ||
*Determine number of stocks that can be made | *Determine number of stocks that can be made | ||
Revision as of 10:39, 9 August 2010
Necessary Labels for All Stocks
- Position on cap insert (e.g., 1A1 for Box 1, position A1)
- Cell line name (e.g., CTLL-2 Parental)
- Passage number (for adherent cells)
- Cell number in the stock (e.g., 5.0 x 10^6 cells)
- Initial and date
Lymphocyte (CTLL-2, Jurkat, and NK-92) Stocks
- Count cells with Trypan Blue
- Determine number of stocks that can be made
- Update freezer stock spreadsheet to determine stock position and print appropriate labels
- Harvest desired number of cells by spinning at 1400 rpm for 10 min
- Resuspend cells with heat-inactivated FBS + 10% DMSO to 10-20 million cells per ml
- Aliquot cells into cryovials at 0.5 ml/vial (i.e., 5-10 million cells per vial)
- Move tubes into Mr. Frosty (make sure the tube rack is dry, sufficient volume of isopropanol is in the container, and isopropanol hasn't gone through more than 5 cycles of freeze/thaw)
- Store Mr. Frosty at -80 degC for 24 hours
- Transfer tubes to appropriate box and position in cryo container
HEK Stocks
- Trypsinize, neutralize, and count cells with Trypan Blue
- Determine number of stocks that can be made
- Update freezer stock spreadsheet to determine stock position and print appropriate labels
- Harvest desired number of cells by spinning at 1400 rpm for 10 min
- Resuspend cells with media + 10% DMSO to 1-2 million cells per ml
- Aliquot cells into cryovials at 1 ml/vial
- Move tubes into Mr. Frosty (make sure the tube rack is dry, sufficient volume of isopropanol is in the container, and isopropanol hasn't gone through more than 5 cycles of freeze/thaw)
- Store Mr. Frosty at -80 degC for 24 hours
- Transfer tubes to appropriate box and position in cryo container
