Viability and apoptosis measured by imaging in BRAF(V600E/D) melanoma cell lines following treatment with combinations of two compounds (viability/apoptosis dataset 1 of 2) - Dataset (ID:20272)

HMS Dataset ID: 20272
Dataset Title: Viability and apoptosis measured by imaging in BRAF(V600E/D) melanoma cell lines following treatment with combinations of two compounds (viability/apoptosis dataset 1 of 2)
Publication(s) Using Dataset: PMID: 28069687
Project Summary Page(s): lincs.hms.harvard.edu/fallahi-sichani-molsystbiol-2017
Screening Lab Investigator: Mohammad Fallahi-Sichani
Screening Principal Investigator: Peter K. Sorger
Assay Description: The dose response of human melanoma cell lines in response to Vemurafenib in combination with a second compound was measured at 72 hr to determine their effects on apoptosis and viability. Cells were treated in 3 replicates using a Hewlett-Packard (HP) D300 Digital Dispenser with 4 doses of Vemurafenib (1:3.16 dilution ratios) and 1 or 3 doses of a second compound for 72 hr. To score viability and apoptosis, a dye-based imaging assay was used. The cell-permeable DNA dye Hoechst 33342 was used to mark all nuclei, and DEVD-NucView488 Caspase-3 substrate was used to mark the nuclei of cells undergoing apoptosis (i.e., in which caspase 3 is active).
Assay Protocol: 1. Cells were seeded at the following densities in 96-well plates (Corning, cat. #3603) in 180 μL of full growth media: MMAC-SF and WM-115, 5,000 cells per well; MZ7-mel, 3,500 cells per well; COLO 858 and A-375, 2,500 cells per well. Cell counts were based on measurements using a Cellometer Auto T4 Cell Viability Counter (Nexcelom Bioscience).
2. After 24 hr of normal growth, cells were treated in 3 replicates using a Hewlett-Packard (HP) D300 Digital Dispenser with 4 doses of Vemurafenib (in 1:3.16 dilution ratios) in combination with 1 or 3 doses of a second compound for 72 hr.
3. To score viability and apoptosis, a dye-based imaging assay was used. The cell-permeable DNA dye Hoechst 33342 was used to mark all nuclei, and DEVD-NucView488 Caspase-3 substrate was used to mark the nuclei of cells undergoing apoptosis (i.e., in which caspase 3 is active). Into each well containing 180 µL of medium 60 µL was dispensed of a cocktail of reagents, including 4 µg/mL Hoechst 33342 (Invitrogen) and 2 µM DEVD-NucView488 Caspase-3 substrate (Biotium) in phosphate-buffered saline (PBS), so that the final concentrations of Hoechst 33342 and NucView488 were 1 µg/mL and 500 nM, respectively.
4. Plates were incubated in a tissue culture incubator (37°C, 5% CO2) for 1.5 hr.
5. To make plate reading less time sensitive, cells were fixed after staining but were not washed before imaging. To each well 26.6 µL of pre-warmed 10% paraformaldehyde in PBS were added (final concentration of 1%). Plates were spun briefly at 1000 rpm while cells were being fixed for a total of 20 min at room temperature.
6. The plates were then sealed using Microseal aluminum foil (Bio-Rad) and were imaged with a 10x objective on an Operetta imaging system (PerkinElmer). Eleven sites were imaged in each well.
7. Image segmentation and analysis were performed using Acapella software (PerkinElmer). Nuclear segmentation with Hoechst 33342 was used to identify individual nuclei and determine the total number of cells. To score apoptotic cells, bright spots were detected by dividing the NucView488 channel nuclear intensity by the nucleus area, and spots brighter than a separating threshold were scored as apoptotic. Relative viability was calculated by subtracting the number of apoptotic cells from the total number of cells at each condition followed by normalization to a DMSO-treated control. The apoptosis fraction was calculated by dividing the number of apoptotic cells by the total number of cells.
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2016-12-22
Most Recent Update: 2017-01-20