Oncolytic Virus Purification from Infected Mammalian Cells

Take an oncolytic virus-infected mammalian cell suspension.

Sonicate to lyse the cells and release the viruses into the solution.

Add DNase and its cofactor, then mix thoroughly and incubate.

The cofactor activates DNase, which degrades host DNA and reduces lysate viscosity.

Centrifuge the lysate to pellet host cell debris.

Collect the supernatant and filter it through a membrane to remove large host contaminants.

Repeat the filtration twice using membranes with progressively smaller pore sizes. This removes smaller contaminants while retaining the viruses in the filtrate.

Carefully layer the filtrate over a dense sucrose solution and centrifuge at high speed.

The viruses migrate to the bottom of the tube, while lighter contaminants remain in the supernatant.

Discard the supernatant and sucrose layer.

Add a stabilizing buffer that preserves viral integrity, then vortex to resuspend the viruses.

The purified viruses are now ready for further studies.

Thaw the previously snapped frozen cells in a warm water bath at 37 degrees Celsius and sonicate for one minute for three cycles in a water bath. Add 175 units of Benzonase Nuclease and two microliters of two millimolar magnesium chloride per milli