Highly multiplexed imaging by Cyclic Immunofluorescence (CycIF) of protein levels and protein phosphorylation states in the COLO 858 melanoma cell line - Dataset (ID:20236)
- Small Molecules Studied
- Cell Lines Studied
- Proteins Studied
- Antibodies Studied
- Other Reagents Studied
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|HMS Dataset ID:||20236|
|Dataset Title:||Highly multiplexed imaging by Cyclic Immunofluorescence (CycIF) of protein levels and protein phosphorylation states in the COLO 858 melanoma cell line|
|Publication(s) Using Dataset:||PMID: 26399630|
|Project Summary Page(s):||lincs.hms.harvard.edu/lin-natcommun-2015|
|Screening Lab Investigator:||Jia-Ren Lin, Mohammad Fallahi-Sichani|
|Screening Principal Investigator:||Peter Sorger|
|Assay Description:||Cyclic Immunofluorescence (CycIF) is a novel fluorescence imaging assay used for multiplexed measurement of protein levels and protein phosphorylation states in single cells. COLO 858 melanoma cells plated in 96-well plates were treated with different doses of PLX4032/Vemurafenib for 48 hr before being fixed and subjected to two cycles of CycIF staining according to the protocol described below.|
1. COLO 858 cells were plated at 20,000 cells per well in duplicate 96-well plates and grown for 24 hours to 70-80% confluency. Cell growth conditions are reported in PMID: 25814555.|
2. The cells then were treated with the indicated doses of PLX4032/Vemurafenib for 48 hrs.
3. After drug treatment, the cells were fixed with 140 ul 4% PFA at room temperature for 30 minutes and then washed 4 times with 250 ul PBS per wash using a Bio-Tek EL406 plate washer.
4. To permeabilize the cells, 140 ul ice-cold methanol were added, and the plates were left at room temperature for 10 minutes. The plates then were washed 4 times with 250 ul PBS per wash as above.
5. The permeabilized cells were blocked by incubation with 50 ul Odyssey blocking buffer at room temperature for 1 hr.
6. 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 4 times with 250 ul PBS per wash using the same Bio-Tek EL406 plate washer on the lowest setting.
7. Hoechst 33342 (1mg/ml stock) was diluted 1:5000 in 140 ul PBS and incubated with the cells for 15 minutes at room temperature, and the cells then were washed gently 4 times with 250 ul PBS as above.
8. To image the fluorescent signals, a Cytell (GE Healthcare Life Sciences) imager was used with a 10x objective lens and fixed filter setting for 4-channel immunofluorescence (Hoechst, 488 nm, 555 nm, and 647 nm) to minimize crosstalk. Weak signals from fluorophore-conjugated primary antibodies required a binning option or high-sensitivity camera (EMCCD, sCMOS).
9. To inactivate the fluorphore(s) used in the first round of imaging, the PBS in each well was removed, 140 ul of fluor-inactivation solution were added, and the plates were incubated at room temperature for 1 hr with light exposure from a tabletop lamp or similar. Each well then was washed gently 4 times with 250 ul PBS using a plate washer as above.
10. After fluor inactivation, the cells were imaged again to ensure complete removal of dye signal.
11. The cells then were subjected to a second round of labeling by repeating the blocking (step 5) through imaging (step 8) steps described above.
12. To align the images from the two sequential rounds of labeling, the two Hoechst images were used as reference images and registered using a rigid body transformation function. The same registration information then was used to align the images from the other fluorescent channels (488 nm, 555 nm, and 647 nm).
13. Image segmentation and analysis were performed using ImageJ and the scripts available at http://lincs.hms.harvard.edu/lin-natcommun-2015/. In brief, the Hoechst images were converted to nuclear masks. Nuclear masks were converted into RING regions of interest (ROIs) and used to define the quantification areas for the images in each of the other fluorescent channels. For fluorescence intensity measurement, the raw images were first background subtracted using the rolling ball method with a 50-pixel radius. Then, the nuclear masks/ROIs were applied to each image and the intensity (reported in arbitrary units) measured. Cell area, perimeter, and circularity also were calculated using default functions in ImageJ. Between 500 and 3500 cells per condition were analyzed, and links to the raw and registered images are provided in the accompanying dataset as well as the x-y coordinates for each analyzed cell in the registered images.
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2015-09-15|
|Most Recent Update:||2016-08-24|