################################################################
Dataset Information
################################################################

Dataset Name:
LINCS MCF 10A Common Project: Fixed-time-point sensitivity measures of the MCF 10A breast cell line to 8 small molecule perturbagens. Dataset 7 of 9: Calculated dose-response metrics for biological replicate 1

Dataset Description:
To generate measures of the sensitivities of three independent batches and one derivative of the MCF 10A cell line to 8 small molecule perturbagens, we treated cells with single drugs over a minimum 9-point dilution series using SQRT(10) dilutions centered around the GR<sub>50</sub> but not exceeding 10 M and then measured cell number after three days of drug exposure.

--Data in Package:
20284.txt

--Metadata in Package:
Small_Molecule_Metadata.txt
Cell_Line_Metadata.txt

################################################################
Center-specific Information
################################################################

Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20284

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20284/

################################################################
Assay Information
################################################################

Assay Protocol:
1. Cells in mid-log phase of the growth cycle for three independent batches of MCF 10A and for one batch of an H2B-mCherry-expressing derivative of MCF 10A were each plated at 750 cells/well in 60 L of complete growth medium (DMEM: F-12 + 5% (v/v) horse serum + 10 g/mL human insulin + 20 ng/mL rhEGF + 100 ng/mL Cholera toxin + 0.5 g/mL Hydrocortisone) in triplicate 384-well plates. <br />
2. The plated cells were grown for 24 hours at 37C in the presence of 5% CO<sub>2</sub>.<br />
3. Two of the plates were treated with the indicated small molecules by direct dispensing of DMSO stock solutions to the indicated concentrations into 60 L of media using an HP D300 Digital Dispenser and then were allowed to grow for 3 additional days.<br />
4. Immediately after treatment of the two plates, the untreated third plate was fixed and stained by adding 20 L of staining solution (1:1000 LIVE/DEAD Far Red Dead Cell Stain (Thermo Fisher Scientific, catalog #L-34974), 2 M Hoechst 33342 (Thermo Fisher Scientific, catalog #62249), 10% OptiPrep (Sigma-Aldrich, catalog #D1556-250ML) in PBS). The plate was incubated for 30 min at room temperature, 90 L of supernatant per well was removed and replaced with the same volume of PBS, and the plate was imaged as described in step 6.<br />
5. On day 3 the treated plates were fixed, stained, and prepped for imaging as described in step 4.<br />
6. The plates were scanned with a PE Operetta high-throughput plate scanner.<br />
7. Nuclei for cells that were alive at the time of fixation were quantified using Columbus software.<br />
8. Nuclei counts were normalized to DMSO-treated controls on the same plate to yield relative cell count and normalized growth rate inhibition (GR) values for each technical replicate for each cell line batch / small molecule / small molecule concentration combination. 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. Consistent with the methods reported in Hafner et al. (2016) (PMID:  <a href="http://www.ncbi.nlm.nih.gov/pubmed/27135972" target = "_blank">27135972</a>), normalized growth rate inhibition (GR) 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 the 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. Data for three complete biological replicates were analyzed (HMS LINCS Datasets #20278-20280).<br />
9. Nuclei counts for all technical replicates within a single biological replicate were averaged to yield mean relative cell counts and the mean normalized growth rate inhibition (GR) value for each cell line batch / small molecule / small molecule concentration combination within each biological replicate (HMS LINCS Datasets #20281-20283).<br />
10. Again within each biological replicate, mean normalized growth rate inhibition (GR) values for a given cell line batch / small molecule combination across all tested concentrations were fitted to a sigmoidal curve to extract the GR<sub>50</sub>, GEC<sub>50</sub>, GR<sub>max</sub>, GR<sub>inf</sub>, Hill coefficient, GR_AOC, and r<sup>2</sup> values (HMS LINCS Datasets #20284-20286).<br />

Date Updated:
2017-03-14

Date Retrieved from Center:
11/13/2015

################################################################
Metadata Information
################################################################

Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
[/generic/datapointFile]
[/generic/reagents_studied]
