Synergy microplate reader

Cells (KMH2, CAL62 and THJ-21T) were seeded at a density of 2,400 cells/well into a 96-well plate and incubated overnight. After a 24 h incubation period, cells were treated with increasing concentrations of Lestaurtinib (0.06 to 4.0 μM) for 72 h. After 72 h, cells were incubated with PrestoBlue for 1 h at 37°C. The same procedure was completed using the normal cell line, WI-38, with increasing concentrations of Lestaurtinib (0.125 to 2.0 μM) for 72 h. For ATC cell lines, two biological replicates were completed with 3 technical replicates for each concentration. For WI-38, one biological replicate was completed with 3 technical replicates for each concentration. Fluorescence readings were completed using a BioTek Synergy Microplate Reader with 560nm excitation and 590nm emission wavelengths. These values were normalized to the untreated controls and the average viability for each concentration was calculated. In order to determine the half-maximal inhibitory concentration (IC₅₀), the normalized relative fluorescence values of the Lestaurtinib-treated samples were calculated as a percentage of the mean relative fluorescence units of the untreated, control samples. Drug concentrations were transformed to a logarithmic scale and IC₅₀ values were calculated using non-linear regression (curve fit). Analysis was completed using Prism 7 Graphpad Software.

The assay was performed in triplicate. PC12 cells were maintained in DMEM containing 5% FCS and 10% HS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO₂ at 37 °C. The tested compounds were dissolved in DMSO, and the final volume for DMSO in each well was no more than 0.1%. To rule out a potential influence of the solvent, we conducted a corresponding control experiments. PC12 cells were seeded in 96-well plate at a density of 5 × 10⁴/well. After cell attachment overnight, each well was treated with different concentrations of test compounds or 0.1% DMSO for 24 h. Then 10 μL of MTT solution (5 mg/mL) was added to each well and the cultures were incubated for another 4 h at 37 °C. The supernatant was then removed and 100 μL of DMSO was added to each well and agitated at 60 rpm for 5 min to dissolve the precipitate. The absorbance was read at 570 nm by a SYNERGY microplate reader (BioTek, Winooski, VT).

MDA-MB-435 cells in log-phase growth were seeded in white-walled, clear-bottomed 96-well microtiter plates and incubated overnight. The next morning, DMSO (vehicle), vinblastine or silvestrol were added to the cells to a final volume of 100 μL. After 24–48 h incubation, caspase 3/7 activity was assessed using a commercial luminescence kit according to the manufacturer’s instructions (Caspase-Glo® 3/7 Assay, Promega Corp.). The Caspase-Glo reagent, which also serves to lyse the cells, was added to each well (100 μL) and the contents were mixed gently and incubated at room temperature for 90 min. The resulting luminescence was measured using a Synergy microplate reader (BioTek Instruments, Winooski, VT).