High-content imaging of six breast cancer cell lines treated with a library of small molecule kinase inhibitors. - Dataset (ID:20244)
|HMS Dataset ID:||20244|
|Dataset Title:||High-content imaging of six breast cancer cell lines treated with a library of small molecule kinase inhibitors.|
|Project Summary Page(s):||lincs.hms.harvard.edu/mills-unpubl-2015|
|Screening Lab Investigator:||Caitlin E. Mills|
|Screening Principal Investigator:||Peter K. Sorger, David W. Andrews|
|Assay Description:||To investigate the phenotypic effects that kinase inhibitors have on breast cancer cell lines at the single cell level, five breast cancer cell lines (hormone receptor-positive MCF7 cells, Her2-amplified SK-BR-3 cells, and triple negative BT-20, MDA-MB-231, and Hs 578T cells) and one non-tumorigenic breast cell line (MCF 10A) were treated at three doses with a panel of 105 kinase inhibitors covering a broad range of targets. A panel of twelve control treatments with small molecule and protein perturbagens known to induce particular types of cell stress/death was included for reference. The cells were stained with DRAQ5 (to visualize nuclei and cytoplasm) and TMRE (an indicator of mitochondrial membrane potential). The experiment was performed in biological triplicate. A subset of the data from one of the three biological replicates is presented here to illustrate some of the cellular features assayed. The complete dataset is available at http://lincs.hms.harvard.edu/mills-unpubl-2015/.|
1. Cell culture conditions: MCF 10A cells were cultured in DMEM/F-12 media supplemented with 5% horse serum, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 20 ng/ml EGF, 100 ng/ml cholera toxin, and 1% penicillin/streptomycin. BT-20 cells were cultured in EMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. MCF7, MDA-MB-231, and Hs 578T cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. SK-BR-3 cells were cultured in McCoy’s media with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were maintained at 37ºC and 5% CO2.|
2. Cells were seeded at 2500-3500 cells per well in 40 µl of media in 384 well plates and allowed to adhere for 24 hours.
3. A 5 mM master compound plate was created in DMSO, and plates containing 5x–concentrated stocks across a dilution series were prepared in 10 mM HEPES.
4. 10 µl of the relevant 5x solution then were transferred to each well of cells in the treatment plates using a 96-channel pipettor for final compound concentrations of 10 µM, 1.11 µM, and 0.12 µM. Cells were incubated in the presence of compound for 24 hours.
5. 10 µl of a 6x-concentrated dye solution in media were added using a 96-channel pipettor to each well of cells in the treatment plates, yielding final concentrations of 10 nM TMRE and 7.5 µM DRAQ5. Cells were incubated in the presence of dye for 30 min.
6. Cells were imaged live using a 40x water immersion objective on a confocal Perkin Elmer Opera microscope. Eight to ten fields of view were acquired per well.
7. Cell segmentation and feature extraction were performed using Acapella software, and the script and parameter files are available at http://lincs.hms.harvard.edu/mills-unpubl-2015/. Cell segmentation was based on the DRAQ5 signal. Quantitative intensity, morphology, and texture features were extracted for the nuclear and cytoplasmic regions. See Haralick et al (1973) and Hamilton et al (2007) for additional details.
8. The entire experiment was performed in biological triplicate. A subset of the data from one of the replicates is presented here to illustrate some of the cellular features assayed. The complete dataset is available at http://lincs.hms.harvard.edu/mills-unpubl-2015/.
|Assay Protocol Reference:||
Haralick, R.M., Shanmugam, K., and Dinstein, I. (1973) Textural features for image classification. IEEE Trans Syst Man Cybern. SMC-3, 610-621.|
Hamilton, N.A. Pantelic, R.S., Hanson, K., and Teasdale, R.D. (2007) Fast automated cell phenotype image classification. BMC Bioinformatics. 8, 110. doi:10.1186/1471-2105-8-110 PMID:17394669 PMCID: PMC1847687
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2015-12-16|
|Most Recent Update:||2016-11-08|