TITLE: Alternative method to visualize receptor dynamics in cell membranes
AUTHORS: Ravelli Cosetta, Corsini Michela, Ventura Anna, Domenichini Mattia, Grillo Elisabetta, Mitola Stefania

[DESCRIPTION]
This protocol details alternative method to visualize receptor dynamics in cell membranes.

[STEPS]
SECTION: Cell transfection (day 1 and 2)
1. The day before transfection, seed 2x104/cm2 of CHO cells in 6 well plate in F12 medium supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin and 10% FCS (complete medium) and culture under normal conditions at37 °C in 5% CO2. When using different cell lines, ensure a cell density to allow 60-70% of confluence the day of the transfection.

SECTION: Cell transfection (day 1 and 2)
2. After 1440 min, transfect each well of CHO cells with 4 µg of pBE-hVEGFR2-eYFP and 8 ngof PEI  (1µg/µL) in serum and antibiotics free F12 medium. 

After 240 min, replace medium with complete medium.

SECTION: Protein immobilization to glass coverslip (day 3)
3. Incubate the 2 well chambered glass coverslips with 100 µL of sterile PBS containing 2 μg/mL of human VEGF-A for 960 min at 4 °C. The coating is carried out by placing a drop containing the recombinant protein in the center of the well in order to obtain a 10 mm diameter coating spot. VEGF-A can be replaced by other ligands able to recruit other specific receptors.
SECTION: Protein immobilization to glass coverslip (day 3)
4. After 960 min, remove unbound ligand and wash the coverslips 3 times with cold and sterile PBS. 
SECTION: Protein immobilization to glass coverslip (day 3)
5. Block nonspecific binding sites with 1 mg/mL of BSA for 60 min.

SECTION: Protein immobilization to glass coverslip (day 3)
4.1. Wash the coverslips  with cold and sterile PBS. (1/3)
SECTION: Protein immobilization to glass coverslip (day 3)
4.2. Wash the coverslips  with cold and sterile PBS. (2/3)
SECTION: Protein immobilization to glass coverslip (day 3)
4.3. Wash the coverslips with cold and sterile PBS. (3/3)

Under these conditions, ligand binds to the coverslip in a dose-dependent manner, with maximal binding at coating concentrations ≥ 2 μg/mL. Using this concentration, it is possible to have a spot with a high concentration of ligands. 
Substratum-immobilized ligand is resistant to high molar salt (2 mol/L NaCl) and detergent (0.2% Triton X-100) washes [1,2].

SECTION: Cell preparation (day 3)
6. Put glass coverslips on the bottom of a 24 well plate and ensure it remains to the bottom of the well while seeding the cells.
SECTION: Cell preparation (day 3)
7. 24 hours after cell transfection, plate CHO at the density of 75.000/cm2 in complete medium on the coverslips and culture under normal conditions for 960 min. When using different cell lines, ensure a cell density to allow 50-80% of confluence the day of image acquisition.
SECTION: Image acquisition (day 4)
8. Replace the complete medium of transfected cells with F12 (without phenol red) 1% FCS and culture under normal conditions for 120 min.
SECTION: Image acquisition (day 4)
9. After 120 min of starvation, flip upside-down the cell-plated coverslips on immobilized-VEGF  chambered in F12 1% FCS.

SECTION: Image acquisition (day 4)
10. Put the sample in the microscope incubator at 37 °C and 5% CO2.

To analyze VEGFR2 recruitment we acquired Z-stack images for 120 min. 

SECTION: Image acquisition (day 4)
11. Acquire images using YFP fluorescence filter set (excitation: 500/20; dichroic: long pass 512; emission: 535/30).
 
 

SECTION: Image acquisition (day 4)
12. Acquire imaging with a PlanApochromat 63X/1.4NA Oil objective and Apotome structured illumination that allow a sectioning of 1.3 µm. Set an overlap of 50% between two consecutive stacks. On average, a whole CHO cell is acquired in 10-12 slices, with a total thickness of acquisition of 13-15.6 µm.
SECTION: Image acquisition (day 4)
13. Process images without deconvolution.
SECTION: Image quantification and data analysis (day 5)
14. Open image series in Fiji as hyperstacks. A sequence of images open, each representing a stack. 
SECTION: Image quantification and data analysis (day 5)
15. Convert image stack in 8 bits.
SECTION: Image quantification and data analysis (day 5)
16. Adjust brightness and contrast in order to clearly see cells in each stack. 

 

SECTION: Image quantification and data analysis (day 5)
17. In Analyze > Set Measurements select Area and Area percentage options.
SECTION: Image quantification and data analysis (day 5)
18. Open the threshold menu and set threshold manually in order to clearly see the specific fluorescence standing out from the background.

 

SECTION: Image quantification and data analysis (day 5)
19. Draw, using freehand selection, the projection of the cell. Analyze one cell at a time. Scroll through the image sequence measuring Area and Area percentage in every stack.
SECTION: Image quantification and data analysis (day 5)
20. Save data for the analysis.
SECTION: Image quantification and data analysis (day 5)
21. Calculate the number of pixels positive for VEGFR2 associated fluorescence using the formula:

Nº of VEGFR2-positive pixels = Area percentage * (Area/100)
SECTION: Image quantification and data analysis (day 5)
22. Sum all the pixel from each Z-stack to obtain the total amount of VEGFR2-positive pixels for cell
SECTION: Image quantification and data analysis (day 5)
23. Calculate the distribution of VEGFR2 in each stack using the formula:

% of VEGFR2 area = ( Nº of VEGFR2-positive pixels / total amount of VEGFR2-positive pixels) * 100

 

SECTION: 3D reconstruction
24. Select a region of interest (ROI) that includes one cell or more. Save the image.
SECTION: 3D reconstruction
25. Create orthogonal projection by choosing, from Image > Stacks the Orthogonal Views command. 
SECTION: 3D reconstruction
26. Create a 3D image using “3D viewer “ plugin.