Multiplexed imaging of protein levels and protein phosphorylation states in the MCF 10A breast cell line treated with EGF - Dataset (ID:20265)
|HMS Dataset ID:||20265|
|Dataset Title:||Multiplexed imaging of protein levels and protein phosphorylation states in the MCF 10A breast cell line treated with EGF|
|Screening Lab Investigator:||Saman Honarnejad|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||The MCF 10A breast cell line was plated in 96-well plates and treated with different doses of epidermal growth factor (EGF) for 24 hr before being fixed and analyzed by immunofluorescence microscopy according to the protocol described below.|
1. MCF 10A cells were plated at 5,000 cells per well in duplicate 96-well plates and grown for 48 hr to 70-80% confluency. Cell growth conditions are reported in PMID: 27135972. Cells were grown under the reported conditions for 24 hr, serum starved overnight, and then treated with the indicated doses of EGF for 24 hr.|
2. After treatment, cells in each well were fixed with 40 uL 2% PFA at room temperature for 10 min and then washed 3 times with 200 uL PBS per wash.
3. To permeabilize the cells, 40 uL ice-cold methanol were added, and the plates were left at room temperature for 10 minutes. The cells were then washed 3 times with 200 uL PBS per wash.
4. The permeabilized cells were blocked by incubation with 50 uL Odyssey blocking buffer at room temperature for 1 hr.
5. Primary antibodies were diluted in 50 uL Odyssey buffer and added to the cells. The cells were incubated overnight at 4°C and then washed gently 3 times with 200 uL PBS per wash. Diluted secondary antibodies were added to the cells, and the cells were incubated for 1 hr at room temperature and washed as just described.
6. Hoechst 33342 (1 mg/mL stock) was diluted 1:5000 in 150 uL PBS and incubated with the cells for 15 minutes at room temperature, and the cells were washed gently 3 times with 200 uL PBS.
7. Whole cell blue dye was diluted (1:1000) in 50 uL PBS and incubated with the cells for 15 minutes at room temperature, and the cells again were washed gently 3 times with 200 uL PBS.
8. To image the fluorescent signals, an Operetta microscope (Perkin Elmer) was used with a 10x objective lens and filter setting for 4-channel immunofluorescence (Hoechst, 488 nm, 568 nm, and 647 nm) to minimize crosstalk.
9. Image segmentation and analysis were performed using the Columbus software package. Nuclear segmentation was based on Hoechst 33342 staining, and cell boundaries were segmented based on whole cell blue dye staining.
10. The complete set of images is viewable online through our OMERO server lincs-omero.hms.harvard.edu/webclient/?show=screen-2302. Lookup tables for antibody information and for data file data column definitions also are contained within the downloadable data package for this dataset.
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2016-09-29|
|Most Recent Update:||2016-09-29|