A subclone of the parental CHO line originated by Puck in 1957. The cells have an absolute requirement for L-proline. The cells have been adapted for growth in Sigma CHO Protein Free Medium (C5467). They have been cryopreserved in Sigma Cell Freezing Medium - Serum Free (C2639).
Culture Medium
Sigma CHO-protein free medium + 4mM Glutamine. Cells should be frozen in either Sigma Cell Freezing Medium - Serum Free (C2639) with 10% DMSO or Sigma CHO Protein Free Medium (C5467) with 7.5%-10% DMSO
Subculture Routine
Viability may be poor on resuscitation and may initially decrease further. Full recovery may take up to 2 weeks. A centrifugation step to remove the cryoprotectant is essential. Rapidly thaw the frozen ampoule in a water bath at 37°C for 1-2 minutes. Transfer the contents to a centrifuge tube and slowly add 5-10ml of pre-warmed growth media. Remove a sample for counting. Centrifuge at 100g for 2-3 minutes to pellet cells and seed at a relatively high density of 5-7 x 105 cells/ml. Leave culture flask upright and observe regularly until viable proliferating cells are seen. Once established use a split ratio of 1:2 approximately every 4 to 5 days; 5% CO2; 37°C.
Other Notes
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