| 方法 | In Vitro Growth Inhibition Assays
For assessment of cell-based potency, cells were plated in 96-well Falcon plates (Becton Dickinson) in the growth media described above. Plating densities that resulted in logarithmic growth of vehicle-treated cells for the duration of the assay were used: HFF, 15,000 cells/cm2; BT474, MCF-7, N87, and CaLu-3, 30,000 cells/cm2; and HN5, A-431, T47D, HB4a, and HB4a c5.2, 10,000 cells/cm2. After 24 h, cells were exposed to compounds at the concentrations indicated in Fig. 2. HFF, BT474, HN5, and N87 cells were treated in low-glucose DMEM containing 5% FBS, 50 μg/ml gentamicin, and 0.3% v/v DMSO. MCF-7 cells were treated in 50% high-glucose DMEM, 50% low-glucose DMEM containing 5% FBS, 50 μg/ml gentamicin, and 0.3% v/v DMSO. T47D, A-431, and CaLu-3 cells were treated in 50% RPMI, 50% low-glucose DMEM containing 5% FBS, 50 μg/ml gentamicin, and 0.3% v/v DMSO. HB4a and HB4a c5.2 cells were treated in 50% DMEM, 50% RPMI 1640 supplemented with 5% FBS, 2.5 μg/ml hydrocortisone, 2.5 μg/ml insulin, 25 μg/ml hygromycin B, 50 μg/ml gentamicin, and 0.3% v/v DMSO. After 3 days, relative cell number was estimated using methylene blue staining. The media were removed, and 100 μl of 0.5% w/v methylene blue dissolved in 50% ethanol and 50% water were added to each well. Plates were washed by immersion in deionized water and allowed to air dry. 1% w/v n-lauroylsarcosine (100 μl) dissolved in PBS was added to each well, and plates were incubated for 30 min at room temperature. The absorbance at 620 nm was read in a Spectra (Tecan) microplate reader. Data were analyzed using curve-fitting macros written for Microsoft Excel. Concentrations with IC50 were interpolated using the method of Levenberg and Marquardt and this equation: y = Vmax × [1 – (xn/(Kn + xn))], where “K” is equal to IC50. |
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