( 4. Transfer 1 ml of t...)The QIAGEN Guide to Animal Cell Culture

4. Transfer 1 ml of the cell suspension (approximately 35 x 106 adherent cells or 510 x 106 suspension cells) into each freezing vial. Label vials with the name of cell line, date, passage number, and growth medium.

Tip - It may also be useful to note the cell density in the freezing vials before storing. This enables determination of the cell density that provides optimal recovery after thawing.

5. Place freezing vials in racks and transfer to a polystyrene box (with walls approximately 15 mm thick) lined with cotton wool. Store box in a 80C freezer overnight.

Tip - It is important that cells are frozen at a rate of 1C/min. A controlled-rate freezing device can be used instead of the polystyrene box and cotton wool method.

6. The next day, quickly transfer the vials to a liquid nitrogen chamber, making sure that the vials do not begin to thaw.

Viability staining

Trypan blue staining provides a method for distinguishing between viable (i.e., capable of growth) and nonviable cells in a culture. This staining method is based on dye exclusion: cells with intact membranes exclude (i.e., do not take up) the dye and are considered viable.

1. Harvest the cells, either by trypsinization (adherent cell cultures) or by centrifugation at 200 x g for 5 min (suspension cell cultures). Resuspend the cells in an appropriate volume of pre-warmed growth medium to give a cell density of at least 106 cells/ml.

2. Add 0.5 ml 0.4% (w/v) trypan blue (see Table 1) and 0.3 ml PBS (see Table 1) or Hanks balanced salt solution (HBSS; see QIAGEN News 2002 No. 3, 22 for composition) to 0.1 ml of the cell suspension. Mix
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