Fluorescence-Based Detection and Purification of Recombinant Vaccinia Virus

Take a microplate containing a culture of fibroblast cells.
Obtain vaccinia virus-infected fibroblast cells. The virus is genetically modified to encode red fluorescent protein (RFP).
Vortex to lyse the cells and release the virus.
Dilute the lysate in media and add it to the wells.
Incubate to allow the virus to bind the fibroblast surface receptors and enter the cells.
The virus replicates inside the cytoplasm to produce new virions.
This causes cell lysis, releasing viruses that spread and infect neighboring cells, creating plaques.
Using fluorescence microscopy, detect RFP in infected cells within each plaque, indicating viral protein expression.
Aspirate the medium and mark the plaque locations.
Using a pipette tip holding a small amount of medium, scrape and aspirate the marked plaques, then transfer to a cryovial.
Repeat to maximize virus recovery.
Store the cryovial at an ultra-low temperature for further analysis.

To begin the first round of purifying the modified VV, seed 15 six-well plates with 300, 000 unmodified CV-1 cells per well. The next day, thaw the frozen suspension of transfected CV-1 cells at 37 degrees Celsius for three minutes, and then vigorously vortex the suspension for 30 seconds to lyse the cells. Then, dilute one microliter of the lysate with three milliliters of DMEM.

Add half a milliliter of this dilution to each plate well of unmodified CV-1 cells. Then, return the plates to the incubator for two days. Two days later, identify RFP-positive plaques using a fluorescence microscope. They can be spotted with a 10X objective.

As they are identified, accurately label the plaques underneath the plate using a marker pen. Now prepare a cryovial containing 200 microliters of serum-free DMEM for each positive plaque to be harvested.

Next, it is important to aspirate all of the medium from the wells with labeled plaques. Then, using a 200 microliter pipette set to aspirate 30 microliters, partially load the pipette with 10 microliters of medium from the cryovial, and then scrape the plaque and draw up the cells with the remaining aspiration. Eject this into the cryovial and repeat this procedure twice more, targeting the same RFP-positive plaque to collect as many cells as possible. Store the collections at negative 80 degrees Celsius.