The overall objective of this experiment is to Fluorescently label dengue virus for microscopic studies. Prepare necessary Buffers and purify dengue virus with sucrose cushion reconstitute the Amy React Alexa flow dye and dengue virus in labeling buffer. Label the dengue virus with a hundred micromolar of Alexa flow.
Die for one hour and stop the labeling reaction with the sub reagent. Purify the label dengue virus through a size exclusion column. Retitrate the labeled virus by plaque assay and estimate the degree of labeling by immunofluorescence assay.
Hi, I'm Sam Jang from the DSO National Laboratories working in Dr.Wee's lab in the program of emerging infectious diseases after NUS graduate medical school. Today I'm gonna show you how to fluorescently labeled Denki virus with Amy Reactive Alexa flu dye for imaging studies. Alexa, flu die, have superior for the stability and are less pH sensitive than common diet such as fluorescein and rumine task, making them ideal for studies on cellular uptake and endosomal transport of the virus.
Alexa flaw labeled dengue virus will facilitate the understanding of dengue pathogenesis. So let's get started. Before the labeling reaction, prepare fresh labeling buffer and stop reagent according to the written protocol and purify dengue virus by sucrose cushion, add appropriate volume of the purified dengue virus to labeling buffer in a micro centrifuge tube to achieve a concentration of 300 million forming units per milliliter.
This can be scaled up proportionately. For batch labeling of virus. Reconstitute the Lyophilized Alexa floor dye to one millimolar in labeling buffer just prior to the labeling reaction.
Minimize exposure to light from this step onwards. For demonstration purpose, the Alexa floor dye will be left unprotected from light 800 microliters of the one millimolar. Alexa flow die to the diluted dengue virus while stirring gently with the micro Pipet tip.
Incubate for one hour at room temperature protected from light mixed by gentle inversions every 15 minutes after one hour, Centrifuge the tubes briefly. An 800 microliter of the sub region while staring gently with the micro pipette incubate for an additional one hour at room temperature. Protected from light makes by gentle inversions every 15 minutes.
In the meantime, it equate the purification column with 25 milliliters of HNE buffer. After the one hour Incubation, apply the labeled virus to the column and start collecting the flow through. Fill the column with HNE buffer.
Once All the labeled virus has entered the matrix, discard the first 2.5 milliliters of flow through and collect the next two milliliters of the labeled virus fraction. Ecor and saw the purified labeled virus at Minus 80 degrees Celsius. Determine the title Of the labeled virus by plaque assay.
In a typical experiment, less than tenfold drop in the virus title should be observed. Post labeling, an immunofluorescence assay should be carried out to evaluate the degree of labeling. To do that enumerate viral cells using a hemo cytometer and appropriate volume of cells to growth media to achieve a density of 50, 000 cells per milliliter.
See one milliliter of viral cells per well in a four well plate containing a sterile glass cover.Sleep. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. Remove the plate from the incubator and aspirate the supinator from well in fact, the cells with approximately one MOI of the labeled virus in a hundred microliters volume for 10 minutes at 37 degrees Celsius.
Remove the Inoculum and wash the cells with 0.5 milliliters of PBS buffer per well. Spirit dis and repeat this procedure two more times. Fix the cells with 200 microliters of 3%per for multi height per well at room temperature for 30 Minutes.
Wash the cells three times with 0.5 milliliters of PBS buffer per well. Alize the cells with ization solution at room temperature for 30 Minutes. Remove the cover sleep from well using fine forceps.
Drain excess fluid by DPing the edge of the cover. Slip against a paper white. Place the cell side of the cover.
Sleep onto 25 microliters of dengue e protein antibody on a piece of parfum. Incubate for one hour in a humid chamber at room temperature protected from light. Next, transfer the cover slip to a six well plate and add two milliliters of wash buffer per oil.
Aspirate the supinate. Repeat these washing Steps two more times after the last wash. Remove the cover slip from well using fine fp.
Drain the excess fluid by dipping the edge of the cover. Slip against a paper wipe. Place the cell side of the cover.
Sleep onto 25 microliters of Alexa floor for eight eight anti mouse secondary antibody on a piece of perfume. Incubate for 45 minutes in a humid chamber at room temperature protected from light after the incubation is completed, transfer the cover sleep to a six Well plate and wash the cells three times with two milliliters of wash buffer For each wash after the last Wash. Remove the cover slip from the well and rinse once in the ionized water.
Drain the excess fluid by dipping against a paper. Wipe mount the cover slip onto a microscope light with eight microliters of mild mounting solution. Allow the mounting solution to set overnight air four degrees Celsius.
The sample is now ready to be visualized with a fluorescent or confocal microscope. Place a small drop of immersion oil on the surface of the cover sleep and fasten the microscope. Slide to the microscope stage.
Adjust the stage until the microscope objective comes into contact with the immersion oil. For a claral image, turn off the ambient lights using the Zes Zen software. Click on the ocular tap for direct observation of the sample through the eyepiece of the microscope.
Find a fill with cells and adjust the focus. Now switch to the acquisition mode. To minimize crosstalk between floral falls, select separate tracks for Alexa floor 4 8 8 and finite four.
Dyes and set acquisitions to sequential excitation and detection. Adjust the laser power gain and offset to ensure that the image is not over or under exposed. Capture the image and analyze an area of interest using colocalization function.
The overlap coefficient can be used to estimate the degree of labeling of dengue virus by the Alexa Flo Dye. A simple immunofluorescence assay was done on viral cells and the degree of labeling can be estimated from the colocalization of the labeled virus With anti e protein, anti antibody staining overlap coefficience range from 0.65 to 0.8 suggesting that approximately 65 to 80%of the variances were labeled with the Alexa floor dye. For the labeling procedure to be successful, it is important to prepare the labeling buffer and start raging fresh and reconstitute the Alexa floor digest prior to the labeling.
The concentration of dye to use can be further optimized to suit your needs. This method of the horizon labeling dengue virus can potentially be adapted to label other viruses. So that's it.
Thanks for watching and good luck for your labeling experiment.
