Cell signaling response to growth factors measured by ELISA - Dataset (ID:20140)

HMS Dataset ID: 20140
Dataset Title: Cell signaling response to growth factors measured by ELISA
Publication(s) Using Dataset: PMID: 24065145; PMID: 24655548
Project Summary Page(s): lincs.hms.harvard.edu/niepel_scisignal_2013; lincs.hms.harvard.edu/niepel-bmcbiol-2014
Screening Lab Investigator: Mario Niepel
Screening Principal Investigator: Peter Sorger
Assay 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 and Akt using ELISA.
Assay Protocol: 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 growth factors (protein ligands) at a final concentration of 1ng/ml and 100ng/ml for 10, 30, or 90 min.
5. The cells were washed in 200 µl of cold PBS and lysed in 30 µl of lysis buffer containing: Mammalian Protein Extraction Buffer (M-PER; Thermo Scientific, 78501) supplemented with protease inhibitor cocktail (Sigma-Aldrich, P2714), 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 5 mM sodium pyrophosphate (Sigma-Aldrich, 221368), 50 µM oxophenylarsine (EMD Biosciences, 521000), and 10 µM bpV (phen; EMD Biosciences, 203695).
6. Plates were sealed, rocked on ice for 15 min, and stored at -80°C.
7. For protein measurements, 384 well, black, flat-bottomed, polystyrene, high-binding ELISA plates (Corning, 3577) were incubated overnight at room temperature with capture antibodies. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
8. Plates were then blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour.
9. Plates were washed four times with 0.05% Tween-20 in PBS (PBS-T) then incubated with lysates and recombinant protein standards for 2 hours at room temperature.
10. After each antibody incubation the plates were washed four times with PBS-T.
11. ELISAs were incubated with primary and secondary antibodies for 2 hours and 1 hour, respectively. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
12. All ELISAs were visualized with SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce, 37069) and luminescent signal was measured with an EnVision Plate Reader (Perkin Elmer).
13. Data from ELISA measurements was extracted using MATLAB Statistical Toolbox.
14. Raw data were background subtracted by subtracting raw values of measuring assay buffer from raw values of each sample measurement.
15. A dilution series of recombinant protein (standard curve) was used to convert raw signal into known protein concentration (pg/ml).
16. A linear best fit line was calculated based on the standard curve, then background subtracted values for each sample were converted into pg/ml using the linear equation (y = mx + b) of the standard curve.
17. Regressed data were multiplied by the dilution factor for each sample, then mean and standard error of the mean values were calculated for at least four technical replicates for the basal profile measurements and two biological replicates for the ligand response measurements.
18. Data above or below the range of detection was set to the upper or lower detection limit, respectively.
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: ELISA
Date Publicly Available: 2013-09-24
Most Recent Update: 2016-11-08