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Vol. 58, No. 4
test samples for 6 and 15 h, respectively, and the cells were centrifuged and
collected. Cell pellets were suspended in 60 ml of an ice cold cell lysis buffer
and incubated on ice for 10 min. This cell pellet suspension was centrifuged
at 10000 g for 5 min, and the supernatant was collected. The cell lysate
(50 ml, equivalent to 200 mg of protein) was mixed with 50 ml of 2ꢅreaction
buffer containing the substrates for caspase-3 (DEVD-pNA (p-nitroanilide)).
After incubation for 2 h at 37 °C, the absorbance at 405 nm of the liberated
chromophore pNA was measured using a microplate reader. The activity of
caspase-3 was evaluated in triplicate.
cells/ml, and 196 ml of this cell suspension was placed in each well of a 96-
well flat-bottom plate. The cells were incubated in 5% CO2/air for 24 h at
37 °C. After incubation, 4 ml of EtOH–H2O (1 : 1) solution containing test
compounds were added to give final concentrations of 0.1—20 mg/ml; 4 ml
of EtOH–H2O (1 : 1) was added into the control wells. The cells were further
incubated for 72 h in the presence of each agent, and then the cell growth
was evaluated by a modified MTT reduction assay. Briefly, after terminating
the cell culture, 10 ml of 5 mg/ml of MTT in PBS was added to every well,
and the plate was reincubated in 5% CO2/air for 4 h at 37 °C. The plate was
then centrifuged at 1500ꢅg for 5 min to precipitate the cells and MTT for-
mazan. An aliquot of 150 ml of the supernatant was removed from each well,
and 175 ml of dimethyl sulfoxide (DMSO) was added to dissolve the MTT
formazan crystals. The plate was mixed on a microshaker for 10 min, and
then read on a microplate reader at 550 nm.
Acknowledgments We are grateful to Dr. Y. Shida and Mr. H. Fukaya,
Tokyo University of Pharmacy and Life Sciences, for the mass spectra and
elemental analyses.
References
A549 Cell Culture Assay A549 cells were maintained in MEM con-
taining 10% FBS in a humidified 5% CO2 atmosphere. Cells were
trypsinized and re-suspended in this medium to 1ꢅ105 cells/ml, and 100 ml
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cell growth was evaluated by a modified MTT reduction assay. The plate
was reincubated in 5% CO2/air for 4 h at 37 °C with MTT 0.2 mg/ml in
MEM supplemented with 10% FBS. After the medium was removed, the
MTT formazan crystals were dissolved with 100 ml of DMSO. The plate was
mixed on a microshaker for 10 min and then read on a microplate reader at
550 nm.
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Matsuo, Yukiko
