This method creates lipid bilayers that mimic an infected cell surface. In order to study the HIV Virological synapse formed with primary human CD four T cells for first label proteins of interest with fluorescent dyes, then create lipid bilayers and allow the cells to form synapses, fix and stain the cells with fluorescent antibodies for visualization of signaling molecules. Then using total internal reflection, fluorescence or turf microscopy acquire images of signaling molecules within the synapse forming cells.
Analyze the turf images to delineate the localization of active signaling molecules with respect to the virological synapse structure. This experimental approach can indicate whether cellular proteins like the lymphocyte specific protein kinase LCK, are recruited to the HIV virological synapse. This method can help address key question in HIV virology and immunology, such as which cell components are involved in the virological synapse.
What is the role in synapse formation and how it may affect the HIV transfer from cell to cell. This bier system provides an endphase view of the synapse with advantage of visualizing the synapse in high special resolution in a single plane. The results clearly the depict the synaptic structure, forming the bier and assembling the flow cell is a kinter, an art form.
Here we demonstrate bilar preparation on the bio optic flow cell for studies using infectious viruses or infected cells or when it's necessary to use small volumes due to limited reagents. A disposable OB flow cells may be used with the same bilar preparation procedure. Michael Kramer and I will demonstrate a procedure For tagging proteins.
Select fluorescent dyes like LOR 4 88 that are effective for binding to proteins are sufficiently photo stable for microscopy imaging and match the excitation wavelengths of the lasers available on the microscope equipment. Start with purified his tagged GP one 20 DH 12 protein. Exchange the protein buffer to sterile PBS using a centrifugal filter unit with molecular weight cutoff of 50 kilodaltons.
Next, check the pH and add sodium bicarbonate pH 9.0 to obtain 50 millimolar final concentration of sodium bicarbonate with a pH of approximately 8.5. Then add a mean reactive LOR 4 88 fluorescent dye at a tenfold molar excess. Incubate this mixture in the dark for 45 minutes to one hour.
Proceed to remove the excess dye with a centrifugal filter unit. Add fresh sterile PBS to the column and continue to wash protein from the free dye. Then concentrate the GP one 20 protein to about one milligram per milliliter.
Measure the concentration of fluorescent dye at 488 nanometers. Also determine the protein concentration with a NanoDrop spectrophotometer. Using the proteins and labels setting.
Divide the two numbers to determine the fluorescence intensity per molecule of protein. Prepare piranha solution of 45 milliliters, sulfuric acid, and 15 milliliters, 30%hydrogen peroxide with plastic clamps. Immerse cover slips for 15 minutes.
Transfer the cover slips to a beaker filled with PIKA purified water Wash each cover slip under running water for two minutes a minute on each side and place on a rack to dry. Proceed to assemble by optics F CS two chambers as previously described by varona etal 2008. Next to capture and present hiss GP one 20 on the bilayer surface without touching the tip to the glass.
Pipette five one microliter drops of liposome mixture onto a micro aqueduct slide To prepare five bilayers per flow cell. Now place a cover slip on top of the lipids. Next, cover the white retaining ring with a stainless steel clamp base unit.
Flip the whole apparatus over and seal. Then mark location of bilayers and incubate for 10 minutes at room temperature. Next, attach tubing with a two-way stop cock to the left perfusion tube.
Fill tubing attached to a three-way stop cock with 1%human serum albumin in heis buffered saline and verify that the setup is devoid of bubbles. Now attach this tubing to the right perfusion tube and generate a positive meniscus. Gently push buffer through the flow cell to block the bilayer.
Attach a one milliliter syringe containing 300 microliters of 100 micromolar nickel chloride ENC casein to the three-way stop cock gently push buffer through close. Both stop cocks before disengaging. The syringe incubate for 30 minutes at room temperature.
Now load 250 molecules of hiss tagged LOR 4 88 labeled GP one 20 per micron to mimic the local density of end clusters found on HIV V one surface. Incubate for 30 minutes in dark at room temperature, wash by layers with five milliliters of H-B-S-H-S-A buffer. Equilibrate the flow cell to 37 degrees Celsius.
Using a one milliliter syringe, add the activated human CD four positive T-cells in 400 microliters of 1%Human serum albumin solution incubate for 45 minutes in a 37 degree Celsius incubator to allow cells attachment to lipid by layer. Next to fix the cells inject 2%paraldehyde, then incubate for 10 minutes at 37 degrees Celsius. Wash three times with one milliliter.
PBS then perme the cells by injecting 0.1%Triton X 100 for five minutes at room temperature after three one milliliter washes with PBS block with casein containing 5%goat serum for 25 minutes. At room temperature wash three times with PBS. Then add primary antibody and 300 microliters buffer per flow cell for 20 minutes to one hour.
At room temperature after three PBS washes, add the appropriate fluorescently labeled secondary antibody in buffer. In the experiment, the control by layer sample is treated with only secondary antibody to determine the level of non-specific signal from this reagent. Incubate for 20 minutes at room temperature, then wash three times with one milliliter.
PBS acquire images of all samples by total internal reflection. Fluorescence microscopy. Perform quantitative analysis of the images with an image analysis application package, such as image J in the analyze set measurements tab.
Check the boxes for area and mean gray value. Select a region of interest on the image that is a polygon or a freehand traced closed shape. Then select the analyze measure tab for the software to record data from this measurement.
In a results table, set the measurements to mean and area then open images for one experimental condition for each cell trace and measure the region of interest and trace and also measure a background region for each experimental condition. Copy measurements and paste into excel. When all data have been collected, calculate the average intensity in arbitrary units.
Subtract background intensity from the mean, then multiply by area to obtain the integrated intensity of the protein in arbitrary units to measure activation and recruitment of the initial membrane proximal signaling molecules. As a result of interaction with GP one 20 at the VIROLOGICAL synapse human primary CD four positive T cells were introduced onto bilayers bearing G one 20 and ICAM one cells introduced to bilayers with only ICAM one served as a control to define the basal levels of signaling. After the cells interacted with bilayers containing GP one 20 and ICAM one LCK protein was recruited to the Virological synapse interface and col localized with GP one 20.
The average intensity of total LCK was higher on the bilayer containing both GP one 20 and ICAM one than with ICAM one alone. Interestingly, the integrated intensity levels were similar. Taken together.
These data suggests that LCK is redistributed into a central cluster. Upon CD four positive T-cell binding to GP one 20. Here the average and integrated phospho LCK tyrosine 3 94 intensities detected by turf microscopy were imaged and quantified.
Results indicate that GP one 20 binding increased phosphorylation of residue tyrosine 3 94 at the LCK activation loop. In contrast, more fin was present in the contact area of cells on ICAM one alone bilayers than on bilayers containing both GP one 20 and ICAM one. Thus, Finn was not recruited to the virological synapse.
Consequently, we conclude that LCK not Finn is the active kinase in the HIV one GP one 20 induced virological synapse. After watching this video, you should have good understanding on how to prefer biers loaded with specific proteins, perform immunofluorescent staining on the flow cell image cell bilar interactions using total internal reflection microscopy, and do basic analysis. This bilar system is also useful for live cell imaging where you can monitor the dynamics of the virological signups formation.
Moreover, cells may be transfected with fluorescently tagged proteins such as GFPs Z 70. This enables tracking of changes in distribution and recruitment of signaling molecules to the synapse in real time.
