Table 1.

Progressive transformation in serial assays with selective killing of focus formers in the 3° assay

Assay	MTX, nM (3–6 day)	2% CS in 1°*	10% CS in 1°*
		% Foci formers
1°	0	0.012	0.02
2°	0	1.4	1.05
3°	0	6.6	5.6
	200	<0.1	<0.1
4°	0 (0)**	18.5	8.4
	0 (200)**	0.006	0.025

10⁵ SA′ cells were seeded in MCDB 402 with 2% or 10% CS in the serial 1° and 2% in all subsequent assays. The 3° assay was of 10³ cells from the 2° assay mixed with 10⁵ A′ cells to form a background for focus formation in 2% CS. MTX (200 nM) was added from days 3 to 6 of the 3° assay. The 2° and 4° assays included cultures with varying numbers of SA′ cells appropriate to make discrete countable numbers of foci. 

^*The 1° assay was in either 2% or 10% CS as indicated. All subsequent assays were in 2% CS. Although the cell layers were thicker in the 1° assay with 10% CS and the individual foci denser than in 2% CS, the foci in subsequent assays, all of which were in 2% CS, were lighter and fuzzier in those originating from the 1° assay in 10% CS than those originating from the 1° assay in 2% CS. 

^**The value (in parentheses) for MTX concentration in the 4° assay refers to that used at 3–6 days of the 3° assay. MTX was not used in the 4° assay.