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The mean population doubling time of the cells was determined as
follows: N=N0ext, where t=time, N0 is the number of cells present at t=0, and
the doubling time is the value of t when N=2N0.
Determination of ICt;n
The IC50 was defined as the concentration of compound necessary to
decrease cell growth to 50% of control growth at a given time. The percentage
of control growth was determined as follows:
[final cell number (treated group) initial inoculum] X 100
final cell number (control group) initial inoculum
The IC50 of MGBG was determined for the first 2 days of treatment.
Cell Viability
Trypan blue (Eastman Kodak, Rochester, NY) was added to 100 pi
aliquots of cells to a final concentration of 0.06%. The cells were mixed well,
and a 10 pi aliquot was transferred to a hemacytometer (Reichert Scientific
Instruments, Buffalo, NY). At least 100 cells per sample were counted with a
phase contrast microscope (Ernst Leitz, Wetzlar, Germany) at 200X
magnification. The percent viability of the cells was determined as follows:
number of cells excluding trypan blue X 100
total number of cells
Clonoaenic Assay
Cells from selected MGBG treatments were counted and diluted to a
concentration of 4 cells/ml. Aliquots of 100 pi were transferred to triplicate 96-
well culture plates (Fisher Scientific) and incubated at 37C for 1 week. The
culture plates were then examined with an inverse phase microscope (Zeiss,
West Germany) for numbers of wells which had colonies of cells. Groups of
>50 ceiis/weil were considered as having been cloned from a single viable