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