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Dataset Information
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Dataset Name:
Cell signaling response to growth factors measured by high throughput microscopy

Dataset Description:
To generate the growth factor signaling profile set, we exposed approximately 40 breast cancer cell lines individually to fifteen different growth factors (protein ligands) for 10, 30, or 90 min. We monitored the response by measuring the phosphorylation state of ERK1/2, Akt, p38, and JNK using high throughput immunofluorescence microscopy.

--Data in Package:
20138.txt

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

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20138

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20138/

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Assay Information
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Assay Protocol:
Antibodies used:<br />
Primary Antibodies:<br />
rabbit--pErk (Cell Signaling Technologies, 4370BC)<br />
mouse--pErk (Cell Signaling Technologies, 9106BC)<br /> 
rabbit--pAkt (Cell Signaling Technologies, 4060BC)<br /> 
mouse--pAkt (Cell Signaling Technologies, 4051BC)<br />
rabbit--pP38 (Cell Signaling Technologies, 4511BC)<br />
mouse--pP38 (Cell Signaling Technologies, 9216BC)<br />
rabbit--pJNK (Cell Signaling Technologies, 9251BC)<br /> 
mouse--pJNK (Cell Signaling Technologies, 9255BC)<br /> 
<br />
Secondary Antibodies:<br /> 
donkey-anti-mouse 488 secondary antibody (Invitrogen #A21202)<br />
donkey-anti-rabbit 647 secondary antibody (Invitrogen #A31573)<br />
<br />
1. Cells were plated either in four 96-well well plates and grown as described in Niepel, Hafner et al. 2013 (PMID: 24065145).<br />
2. Cells were grown for 24 hours and then serum starved in base media without additives for an additional 18 hours.<br />
3. Cell numbers were chosen to yield approximately 75% confluency at the time of ligand treatment.<br />
4. Cells were treated with growth factors (protein ligands) at a final concentration of 1ng/ml and 100ng/ml for 10, 30, or 90 min.<br />
5. The cells were fixed for 10 min at 25C in 2% paraformaldehyde (Electron Microscopy Sciences, #15710).<br />
6. Plates were washed with 200 l PBS-T and stored at 4C until assaying.<br />
7. Cells were permeabilized with 100 l of methanol for 10 min at 25C.<br />
8. Cells were washed with 200 l PBS-T.<br />
9. Cells were blocked with 40 l of Odyssey blocking buffer (OBB; LICOR) for 1 hour at 25C.<br />
10. Cells were treated with 40 l of primary antibody diluted 1:400 in OBB, sealed, and incubate overnight at 4C on rocking platform. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).<br />
11. Cells were washed twice with 200 l of PBS-T.<br />
12. Cells were treated with 40 l of secondary antibody diluted 1:2000 in OBB incubated for 1 hour at 25C. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).<br />
13. Cells were washed in 200 l of PBS-T followed by 200 l of PBS.<br />
14. Cells were stained with 40 l of 250 ng/ml Hoechst 33342 (Invitrogen) and 1:1000 Whole Cell Stain (blue; Thermo Scientific) in PBS.<br />
15. Cells were washed two times with 200 l of PBS and imaged in an imageWoRx high-throughput microscope (Applied Precision). Details about image capture: 3 wavelengths captured per well (excitation at Hoechst 377 nm, GFP 488 nm, near red 647 nm); 4 sites imaged per well for each wavelength. <br />
16. Data was extracted using ImageRail (Millard et al. PMC3105758). Nuclei and cells are counted. Average per cell intensity at 488 nm and at 647 nm were recorded for each well, however only the 647 nm data were used in analysis of this dataset and are therefore only presented here.<br />

Date Updated:
2016-11-08

Date Retrieved from Center:
11/13/2015

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Metadata Information
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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/
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