Cell signaling response to cytokines measured by high throughput microscopy - Dataset (ID:20139)

HMS Dataset ID: 20139
Dataset Title: Cell signaling response to cytokines measured by high throughput microscopy
Publication(s) Using Dataset: PMID: 24065145
Project Summary Page(s): lincs.hms.harvard.edu/niepel_scisignal_2013
Screening Lab Investigator: Mario Niepel
Screening Principal Investigator: Peter Sorger
Assay Description: To generate the cytokine “signaling profile” set, we exposed approximately 40 breast cancer cell lines individually to seven different cytokines (protein ligands or LPS) for 10, 30, or 90 min. We monitored the response by measuring the phosphorylation state of ERK1/2, STAT1, and STAT3 and the nuclear translocation of NF-kappaB using high throughput immunofluorescence microscopy.
Assay Protocol: Antibodies used:
Primary Antibodies:
rabbit-α-STAT1 (Cell Signaling Technologies, 9167BC)
rabbit-α-STAT3 (Cell Signaling Technologies, 9145BC)
mouse-α-NF-κB (Santa Cruz Biotechnology, sc-8008)
mouse-α-pErk (Cell Signaling Technologies, 9106BC)

Secondary Antibodies:
donkey-anti-mouse 488 secondary antibody (Invitrogen #A21202)
donkey-anti-rabbit 488 secondary antibody (Invitrogen #A21206)
donkey-anti-mouse 647 secondary antibody (Invitrogen #A31571)
donkey-anti-rabbit 647 secondary antibody (Invitrogen #A31573)

1. Cells were plated either in four 96-well well plates and grown as described in Niepel, Hafner et al. 2013 (PMID: 24065145).
2. Cells were grown for 24 hours and then serum starved in base media without additives for an additional 18 hours.
3. Cell numbers were chosen to yield approximately 75% confluency at the time of ligand treatment.
4. Cells were treated with cytokines (protein ligands or LPS) at a final concentration of 1ng/ml and 100ng/ml for 10, 30, or 90 min.
5. The cells were fixed for 10 min at 25°C in 2% paraformaldehyde (Electron Microscopy Sciences, #15710).
6. Plates were washed with 200 µl PBS-T and stored at 4°C until assaying.
7. Cells were permeabilized with 100 μl of methanol for 10 min at 25°C.
8. Cells were washed with 200 μl PBS-T.
9. Cells were blocked with 40 μl of Odyssey blocking buffer (OBB; LICOR) for 1 hour at 25°C.
10. Cells were treated with 40 μl of primary antibody diluted 1:400 in OBB, sealed, and incubate overnight at 4°C on rocking platform. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
11. Cells were washed twice with 200 μl of PBS-T.
12. Cells were treated with 40 μl of secondary antibody diluted 1:2000 in OBB incubated for 1 hour at 25°C. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
13. Cells were washed in 200 μl of PBS-T followed by 200 μl of PBS.
14. Cells were stained with 40 µl of 250 ng/ml Hoechst 33342 (Invitrogen) and 1:1000 Whole Cell Stain (blue; Thermo Scientific) in PBS.
15. Cells were washed two times with 200 μl 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.
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.
Assay Protocol Reference: Niepel M, Hafner M, Pace EA, Chung M, Chai DH, Zhou L, Schoeberl B, Sorger PK. Profiles of basal and stimulated receptor signaling networks predict drug response in breast cancer lines..Sci Signal. 2013 Sep 24;6(294):ra84. doi: 10.1126/scisignal.2004379. PMID: 24065145
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2013-09-24
Most Recent Update: 2016-11-08