LINCS Pilot Phase Joint Project: Sensitivity measures of six breast cancer cell lines to a library of small molecule kinase inhibitors (drug combination treatments). Dataset 2 of 2: Mean cell count and mean normalized growth rate inhibition values across technical replicates. - Dataset (ID:20260)
|HMS Dataset ID:||20260|
|Dataset Title:||LINCS Pilot Phase Joint Project: Sensitivity measures of six breast cancer cell lines to a library of small molecule kinase inhibitors (drug combination treatments). Dataset 2 of 2: Mean cell count and mean normalized growth rate inhibition values across technical replicates.|
|Screening Lab Investigator:||Marc Hafner, Mario Niepel|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||As described in HMS LINCS Datasets #20245-20252, measures of the sensitivities of six cell lines to a library of small molecule inhibitors were generated by treating cells with single drugs at six doses starting at 10 µM in 3x dilution steps and measuring cell number after three days of drug exposure. Based on those results, a subset of the drugs was chosen for analysis of combinatorial drug sensitivities in a single cell line (Datasets #20259 and #20260). As part of this LINCS Pilot Phase Joint Project, L1000 gene expression data generated by the Broad LINCS Center for the same perturbagen combinations are available at http://projects.lincscloud.org/#LJP.|
1. BT-20 cells were plated at 2000 cells per well in 60 µL of growth media in quadruplicate 384-well plates. The cells then were grown for 24 hours. For growth conditions, see PMC3845839.|
2. Three plates were treated with the indicated doses and combinations of drugs by direct dispensing of DMSO stock solutions into the appropriate wells of cells using an HP D300 Digital Dispenser.
3. Immediately after treatment of these three plates, the untreated fourth plate for each cell line was fixed and stained by adding 20 μL of fixative containing formaldehyde solution and Hoechst 33342 at a final concentration of 3% formaldehyde and 250 ng/ml Hoechst. This plate was sealed and kept at 4°C until day 3 of the assay.
4. On day 3 the treated plates were fixed, stained, and sealed as described in step 3.
5. After 1 hour all plates were scanned with a PE Operetta high-throughput plate scanner.
6. Nuclei were counted using Columbus software.
7. Nuclei counts were normalized to DMSO-treated controls on the same plate to yield relative cell count and normalized growth rate inhibition values for each plate (technical replicate) for each drug combination (HMS LINCS Datasets #20259). Relative cell count is the measured cell count for a given treatment divided by the 50%-trimmed mean of the cell count of the DMSO-treated control wells on the same plate. Normalized growth rate inhibition values were calculated according to the following formula: 2^[log2(x(c)/x0)/log2(xctrl/x0)]-1 where x(c) is the measured cell count after a given treatment, x0 is the 50%-trimmed mean of the cell count from a day 0 untreated plate grown in parallel until the time of treatment, and xctrl is the 50%-trimmed mean of the cell count of the DMSO-treated control wells on the same treated plate.
8. Second, within each experiment, the results of three plates (technical replicates) were averaged to yield the mean relative cell count and the mean normalized growth rate inhibition value for each cell line and drug combination for a given biological replicate (HMS LINCS Datasets #20260).
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2016-04-01|
|Most Recent Update:||2016-09-09|