LINCS Pilot Phase Joint Project: Sensitivity measures of six breast cancer cell lines to a library of small molecule kinase inhibitors (single drug treatments). Dataset 9 of 15: Cell count and normalized growth rate inhibition values within biological replicate 2. - Dataset (ID:20246)

HMS Dataset ID: 20246
Dataset Title: LINCS Pilot Phase Joint Project: Sensitivity measures of six breast cancer cell lines to a library of small molecule kinase inhibitors (single drug treatments). Dataset 9 of 15: Cell count and normalized growth rate inhibition values within biological replicate 2.
Screening Lab Investigator: Marc Hafner, Mario Niepel
Screening Principal Investigator: Peter K. Sorger
Assay Description: To generate measures of the sensitivities of six cell lines to a library of small molecule inhibitors, we treated cells with single drugs at six doses starting at 10 µM in 3x dilution steps and measured cell number after three days of drug exposure. As part of this LINCS Pilot Phase Joint Project, L1000 gene expression data generated by the Broad LINCS Center for the same set of cells and perturbagens is available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70138.
Assay Protocol: 1. For each cell line except Hs 578T, cells were plated at 2000 cells per well in 60 µL of growth media in quadruplicate 384-well plates per cell line; for Hs 578T, 1000 cells per well were plated. The cells then were grown for 24 hours. For growth conditions, see PMC3845839.
2. Three plates per cell line were treated by transferring 60 nL of 1000x stock solutions of each drug from library plates C-5001-01-LM6-004 (LJP005) and C-5001-01-LM6-001 (LJP006) into 60 µL of media.
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, relative growth, and normalized growth rate inhibition values for each plate (technical replicate) of each cell line at each drug and concentration (HMS LINCS Datasets #20237-20239 and #20245-20247). 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. Relative growth was calculated according to the following formula: (x(c)-x0)/(xctrl-x0) 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. 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, mean relative growth rate, and the mean normalized growth rate inhibition value for each cell line, drug, and concentration for a given biological replicate (HMS LINCS Datasets #20240-20242 and #20248-20250).
9. The mean relative cell count, mean relative growth rate, and mean normalized growth rate inhibition values for each biological replicate then were averaged across the three biological replicates to yield the overall mean relative cell count, mean relative growth rate, and mean normalized growth rate inhibition values and their standard error of the mean (SEM) values reported in HMS LINCS Datasets #20243 and #20251.
10. The overall mean normalized growth rate inhibition values from HMS LINCS Dataset #20251 were averaged across all six concentrations for a given drug and cell line to yield the area over the dose-response curve (GR_AOC) (HMS LINCS Dataset #20252).
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2015-12-18
Most Recent Update: 2017-03-14