Viability and apoptosis in BRAF(V600E/D) melanoma cell lines monitored by imaging - Dataset (ID:20217)
|HMS Dataset ID:||20217|
|Dataset Title:||Viability and apoptosis in BRAF(V600E/D) melanoma cell lines monitored by imaging|
|Publication(s) Using Dataset:||PMID: 25814555|
|Project Summary Page(s):||lincs.hms.harvard.edu/fallahi-sichani-molsystbiol-2015|
|Screening Lab Investigator:||Mohammad Fallahi-Sichani|
|Screening Principal Investigator:||Peter Sorger|
|Assay Description:||Dose response of a set of anti-cancer compounds, including five RAF/MEK inhibitors, in human melanoma cell lines at 24, 48, and 72 hours to determine their effects on apoptosis and viability. Cells were treated in 4 replicates using Hewlett-Packard (HP) D300 Digital Dispenser with either seven or nine doses (in 1:3.16 or 1:2.5 dilution ratios, respectively) of each compound for 24, 48 and 72 h. To score viability and apoptosis, a dye-based imaging assay was used; the cell-permeable DNA dye Hoechst 33342 was used to mark nuclei and DEVD-NucView488 caspase-3 substrate was used to mark apoptosis (stains nuclei of cells undergoing apoptosis, in which caspase 3 is active). The data from this dataset and from the related HMS dataset #20218 were used to generate the signatures presented in HMS datasets #20229-20231.|
1.Cells were seeded at the following densities in 96-well plates (Corning) and grown in full growth media: C32, MMACSF and WM115 (5000 cells per well), MZ7MEL, RVH421, WM1552C, SKMEL28 and K2 (3500 cells per well), COLO858 and LOXIMVI (2500 cells per well).|
2. After 24 hr of normal growth, cells were treated in 4 replicates using Hewlett-Packard (HP) D300 Digital Dispenser with either seven or nine doses (in 1:3.16 or 1:2.5 dilution ratios, respectively) of each compound for 24, 48 and 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 nuclei and DEVD-NucView488 caspase-3 substrate was used to mark apoptosis. 60 µl 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) was dispensed into each well containing 180 µl of medium, 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. 26.6 µl of pre-warmed 10% paraformaldehyde in PBS was added to each well (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. Plates were then sealed using Microseal aluminum foil (Bio-Rad) and were imaged with a 10× objective on Operetta (PerkinElmer). 9 to11 sites were imaged in each well.
7. Image segmentation and analysis were performed using Acapella software (PerkinElmer). The nuclear segmentation with Hoechst 33342 was used to identify individual nuclei and score relative viability. To score apoptotic cells, bright spots were detected by dividing 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. Apoptosis fraction was calculated by dividing the number of apoptotic cells by the total number of nuclei.
|Assay Protocol Reference:||Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J. 2008 Jul;22(7):2243-52. PMID: 18263700|
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2015-01-23|
|Most Recent Update:||2016-10-28|