Selection of mitotic cells

Cells growing as a monolayer round up when they divide. They therefore are less firmly attached to the substratum at this time and may be easily detached (Terasima and Tolmach, 1961, 1963 Robbins and Marcus, 1964 Peterson et al., 1968 Shall, 1973a). 

Exponentially growing cells are used and it is important to maintain constant pH and temperature throughout the selection procedure. Klevecz et al. (1974, 1975) have used CHO cells growing in McCoy s medium with 20% foetal bovine serum and Hepes buffer. They maintain the cells throughout in this medium at 37°C and, of the cells they select, 98-99% are in mitosis. The viability of these selected cells approaches 100% and on reseeding half the cells attach within 1 h of selection and maximum attachment is found by 4 h (Klevecz, 1975). However, they select a very small proportion of the original cells. 

As different cells attach with differing firmness to the substratum, the following procedures may need to be modified for each cell type. 

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This method allows the separation of moderately large cell numbers but sedimentation under unit gravity is a lengthy process and difficulties can be found in keeping the system aseptic. Furthermore, only moderate synchrony is achieved compared with that obtained by selection of mitotic cells. 

This method gives results very comparable to selection of mitotic cells in that almost 100% of the population can be obtained in Gl. It is much simpler than mitotic selection and is more readily applied to larger numbers of cells. 

The selection of mitotic cells has been described in 11.2 and the proportion of cells in mitosis can be increased by use of the mitotic blocking agents colcemid or preferably nocodazole. The combination of a single thymidine block for 15-16 h followed by a 4-5 h in 0.04/ig/ml nocodazole gives a yield of 25-30% when mitotic cells are harvested (Zieve et al., 1980) and further mitotic cells can be obtained with subsequent shakings. 

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