The cover slips used in this assay are functionalized with dextrin then incorporated into microfluidic flow cells. Next, each flow cell is incubated with a suspension of liposomes, which fuse to form a lipid bilayer atop the hydrated dextrin. Under the fluorescence microscope, fluoro four labeled virus and fluorescein labeled streptavidin are added and the virus is allowed to dock to the bilayer.
An influx of acidic buffer initiates viral fusion with the bilayer and a digital camera records the changes in fluorescence throughout the assay. Hi, I'm Daniel Floyd and I'm in the laboratory of Anine Vanian in the Department of Biological Chemistry and Molecular Pharmacology at Harvard University. Today I'm gonna show you a method for visualizing viral fusion kinetics at the single particle level.
We use this procedure in the lab to study the way the virus is getting to host cells. So let's get started. Begin this protocol by preparing the dextrin functionalized cover slips.
The first step is to sonicate the cover slips in solutions of 10%detergent, one molar potassium hydroxide acetone, and 100%ethanol sonicate for 30 minutes per solution. And rinse with deionized water between ations. After the last sonication, rinse the cover slips in water, then dry them in the 80 degrees Celsius incubator.
Next, put the cover slips in an oxygen plasma stripper for three minutes. This step oxidizes the surface of the slides, making the glass uniformly hydrophilic and reactive in the following siloization steps. Next, immerse the cover slips in a fresh solution of 0.2%gly oxy propyl trimeth oxypro trim methoxy in isopropanol and incubate for five minutes.
Then rinse the cover slips several times with isopropanol. Place the Slan coated cover slips at 80 degrees Celsius for one hour. To cure this step allows the slan to covalently bond to the glass.
After curing, arrange the cover slips flat on the inside of a plastic freezer box. Cover the top surface of each cover slip with about one milliliter of a dextrin solution. 30%weight per volume in water.
Close the freezer box and leave the cover slips in a quiet place for 24 to 36 hours. The next day pour off the excess dextrin, grasping the cover slips with plastic forceps and put them back into the ceramic rack. Rinse them with water several times and let them soak in water for 48 hours.
The container may be gently agitated on a tabletop rotator dry the cover slips in an oven set to 80 degrees Celsius and store in a vacuum desiccate until you are ready for the next step. Coating the cover slips with a mixture of liposomes to form a planer bilayer. The next step is to prepare the liposomes to form the lipid bilayer in the hood, combine all of the lipids dissolved in chloroform in a glass test tube.
The liposome formulation includes dia ganglia side, which binds virus particles and a biotinylated lipid, which binds the streptavidin conjugated pH indicator fluorescein. Next, evaporate the solvent under a stream of argon or nitrogen gas. And remove the remaining solvent by leaving the test tube in a vacuum desiccate for two hours.
Re suspend the dried lipid film in 400 microliters HNE buffer. Then transfer the suspension to a plastic micro centrifuge tube. Freeze the suspension in a liquid nitrogen bath and thaw in a warm water bath.
Repeat this freeze thaw cycle four times after the freeze thaw cycles. Use a lipid extruder fitted with a 100 nanometer membrane filter to extrude the lipid suspension. 25 times after extrusion, the suspension should appear to be more transparent.
Store the liposomes at room temperature and use the day of preparation. Now that the lipids are ready to go, prepare the microfluidic flow cell. First, take a 20 by 20 by three millimeter quartz slide and use a diamond burr to drill two one millimeter holes on opposite sides.
Cut a 20 millimeter square from a sheet of double stick tape and using a razor blade. Cut out a 15 by two millimeter section from the center. Peel off one side of the tape backing and adhere the tape to the court slide.
Adhere the other side to a functionalized cover slip now cut to 20 centimeter lengths of polyethylene tubing and insert them into the holes in the quartz slide. Finally, seal the flow cell with five minute epoxy glue. Spread the glue around the sides of the quartz slide to bond it to the cover slip.
Use two more drops of glue to seal the connection between the tubing and the quartz slide. When the glue has cured, prime the flow cell and tubing with HNE buffer. Next to create the supported lipid bilayer, take the extruded liposome suspension and draw about 80 microliters into the channels on the day prior to the fusion experiment.
Begin labeling the virus particles to label the viral interior red. Combine 20 microliters of freshly prepared sulfur rumine or SRB solution with 10 microliters of the viral suspension and leave at room temperature for 20 hours. The next day, remove the excess dye by passing the virus suspension through a PD 10 desalting column and diluting with 800 microliters of HNE buffer.
The column should have an obvious colored band, which contains the labeled virus. Collect this fraction during the ellucian step next, label the viral envelope green by adding 13 microliters of Octa decal, RUMINE one 10 or RH one 10 C 18 to the SRB labeled viral suspension. Stir the suspension by attaching the tube to a laboratory rotator and leave for three hours.
Next, remove excess RH one 10 C 18 using a PD 10 desalting column. As before now the virus is ready for the fusion assay. Perform the fusion kinetics assay using a fluorescence microscope equipped with a high numerical aperture oil immersion objective.
An Argonne krypton laser excites the fluoro fours in the viral membrane and interior. A dichroic mirror then splits the red and green fluorescence emissions and independently focuses the images onto separate halves of an electron multiplying CCD or charge coupled device camera. Mount the cover slip on the microscope stage and attach the outlet tubing to a syringe pump set to flow at 100 microliters per minute.
Clear the flow channels of excess liposomes by flowing in 300 microliters of HNE. Focus the microscope and adjust the laser power to illuminate the surface with 350 watts per centimeter squared of 488 nanometer light to excite the red flora, four and 70 watts per centimeter squared of 568 nanometer light to excite the green flora.Four. Now that the microscope is ready, pump labeled virus diluted one to a hundred into the flow cell.
As the virus diffuses to the bottom surface of the flow channel particles begin to dock. Sticking to the DIA ganglia side, incorporated into the cover slips, planar bilayer. When the density of docked particles reaches about several hundred particles per field of view, begin to flow in a solution of two micrograms per milliliter.
Fluorescein labeled strep streptavidin, a pH sensitive fluorophore as the labeled streptavidin binds to biotin coupled lipid molecules in the bilayer. A dim green background appears again, wash the flow channel with 300 microliters of HNE. Now that the viruses have docked and the bilayer surface is coated with fluorescein, it is time to begin the assay.
Choose an imaging area, focus the microscope and prepare the CCD camera for time lapsed image acquisition. Set the exposure time to 100 milliseconds and collect frames at a rate of 10 hertz. Initiate virus membrane fusion by flowing in an acidic sodium citrate buffer.
As the pH drops the fluorescein fluorescence dims pinpointing the time of the acidification. The CCD camera records the data throughout the assay, which one can use to determine time of hemi fusion and poor formation as documented by the CCD camera. After the pH drop, there are bursts of green fluorescence as the viral and planer membranes fuse, the lipids mix and the green dye in the viral envelope is diluted in the planar membrane.
In contrast, there is a decay in the red fluorescence after the pH drop as the viral and lipid membranes form pores allowing the red dye from the viral interior to escape into the aqueous space below the planar bilayer and out of view, these trends can also be visualized by fluorescent snapshots before and during fusion. Also graph the fluorescence intensity trajectories for each particle as shown in this example, Hemi fusion is defined as the point at which the slope of green fluorescence intensity is at its steepest pore formation as the point at which the red fluorescence decay occurs. Combined data from multiple experiments to generate histograms of hemi fusion and pore formation lag times as shown here, using the time of the pH drop as t equals zero to synchronize data from different experiments.
I've just shown you a way to measure viral fusion kinetics at the single particle level. When doing this procedure. Make sure you use clean cover slips so that you have a homogeneous and fluid bilayer.
Also, try to minimize the amount of light you use during fluorescence imaging so that you can minimize photo damage. So that's it. Thanks for watching and good luck in your experiments.
