Raising the strength of culture medium from 149 or 165 mM by the addition of concentrated saline solutions to about 225-235 mM arrests HeLa cells for 3 h in metaphase without preventing the entry of G2 cells into mitosis. Apart from an initial period of 30 min, metaphases have well developed equatorial plates and chromosomes are discrete from one another.
By employing a standardized continuous wash procedure for detachment of dividing cells during exposure of HeLa monolayers to hypertonicity, excellent yields of mitotic cells consisting almost entirely of metaphases were obtained. They compared favourably with a colcemid control and were superior in yield and synchrony to isotonic collection of mitotic cells. This method is compared with a manual shake-off procedure. Reversibility of the hypertonically collected metaphases by dilution allowed excellent synchronous progression of cells out of mitosis and into their next division 24 h later. Recovery was also studied by conventional plating and growth-kinetic studies to establish the loss of viability after exposure of cells to varying degrees of hypertonicity.
It is concluded that the arrest of cells in metaphase by hypertonicity and the ease of detachment of arrested cells from monolayers offers an excellent synchronizing procedure for HeLa cells.