Smolke:Protocols/Freezer stock management and database

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

Freezer Stock Database

• 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