Smolke:Protocols/Freezer stock management and database

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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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Revision as of 13:38, 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
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