Alternative method to visualize receptor dynamics in cell membranes

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.

After 24h 0m 0s, transfect each well of CHO cells with 4µg of pBE-hVEGFR2-eYFP and 8ngof PEI (1µg/µL) in serum and antibiotics free F12 medium.

• After 4h 0m 0s, replace medium with complete medium.

Incubate the 2 well chambered glass coverslips with 100µL of sterile PBS containing 2 μg/mL of human VEGF-A for 16h 0m 0s 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.

After 16h 0m 0s, remove unbound ligand and wash the coverslips 3 times with cold and sterile PBS.

Wash the coverslips with cold and sterile PBS. (1/3)

Wash the coverslips with cold and sterile PBS. (2/3)

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].

Block nonspecific binding sites with 1 mg/mL of BSA for 1h 0m 0s.

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.

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 16h 0m 0s. When using different cell lines, ensure a cell density to allow 50-80% of confluence the day of image acquisition.

Replace the complete medium of transfected cells with F12 (without phenol red) 1% FCS and culture under normal conditions for 2h 0m 0s.

After 2h 0m 0s of starvation, flip upside-down the cell-plated coverslips on immobilized-VEGF chambered in F12 1% FCS.

Put the sample in the microscope incubator at 37°C and 5% CO₂.

• To analyze VEGFR2 recruitment we acquired Z-stack images for 2h 0m 0s.

Acquire images using YFP fluorescence filter set (excitation: 500/20; dichroic: long pass 512; emission: 535/30).

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.

Process images without deconvolution.

Open image series in Fiji as hyperstacks. A sequence of images open, each representing a stack.

Convert image stack in 8 bits.

Adjust brightness and contrast in order to clearly see cells in each stack.

In Analyze > Set Measurements select Area and Area percentage options.

Open the threshold menu and set threshold manually in order to clearly see the specific fluorescence standing out from the background.

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.

Save data for the analysis.

Calculate the number of pixels positive for VEGFR2 associated fluorescence using the formula:

Nº of VEGFR2-positive pixels = Area percentage * (Area/100)

Sum all the pixel from each Z-stack to obtain the total amount of VEGFR2-positive pixels for cell

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

Select a region of interest (ROI) that includes one cell or more. Save the image.

Create orthogonal projection by choosing, from Image > Stacks the Orthogonal Views command.

Create a 3D image using “3D viewer “ plugin.