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Cryovials, 1.8 ml (Nunc or equivalent)
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Cryovial rack (Nunc or equivalent)
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Sterile Centrifuge tubes, 50 ml (VWR # 21008-146 or equivalent)
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Fetal Bovine Serum, heat inactivated (BioWhittaker # 14-503F or equivalent)
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Sterile DMSO (Sigma # D2650 or equivalent)
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Sterile Pipets of appropriate sizes
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Ice
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Permanent Marking Pen
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Lab Coat
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Latex Gloves
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70 % alcohol or equivalent
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Trypan Blue, 0.4% (GIBCO # 630-5250AG or equivalent)
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Hemocytometer
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Nunc Freezing Container (# 5100-0001)
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Freezer Log
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The Master Cell Bank consists of 20 vials of cells at 5 x 106 cells/vial.
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Before preparing the Master Cell Bank, ensure that the cell line is stabilized and that the cells are free from mycoplasma contamination.
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The day before freezing, refeed the cells with fresh medium, or add additional medium to suspension cultures to ensure that they are in log phase of growth.
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Chill the Freezing medium on ice.
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Label with cryotubes with the cell line name, the cells/vial, and the date. Include on the label that the vials are the Master Cell Bank. Place the vials in the cryotube rack and place the rack on ice.
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Prepare a single cell suspension, use trypsin for adherent cell line.
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Take an aliquot for a cell count. Count the cells using a hemocytometer, according to the current revision of SP 05-009. Determine the viability using trypan blue stain, according to the current revision of SP 09-005. NOTE: The cells must have greater than 90% viability before proceeding. If the cells are less than 90% viability, do not freeze.
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Transfer the cell suspension to centrifuge tubes.
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Centrifuge at 1000 rpm for 5 minutes, 2-8o C.
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Siphon off all the medium.
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Slowly add chilled freezing medium to yield 5 x 106 cells/ml. Resuspend the cells in the freezing medium by gently pipetting.
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Place the tube on ice. Dispense the cells, 1 ml/vial, into the labeled cryovials. Recap vials tightly. Complete all vials.
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Place the vials into the Nunc Freezing Container or Rate Controlled Freezer Racks.
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Replace the lid on the freezing container. Place the Freezing Container in the -700 C Freezer overnight.
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Transfer the vials to the vapor phase of the liquid nitrogen freezer.
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Record the cell line information and location in the Freezer Log.