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Answer Posted / swapna
Plaque Assay Method For adenovirus :
Plate out 293 spinner cells onto 60mm Petri dishes allow to
grow till approx 70% confluent.
It is important to get the cell monolayer as even across the
plate as possible.
Any swirling will result in a high density at the middle and
few cells at the edge.
Make up serial dilution?s of the virus stock. Usually 10-3
to 10-9 this should be done in duplicate.
Remove the media from the cells it is best to do this with
the fan off as the hoods can dry the cells very quickly and
do no more than 6 plates at one time.
Replace the media with 100ul of the appropriate virus
dilution or media for mock infection. Return to the
incubator immediately and leave for 1 hr.
During this time make up enough 50ml aliquots of the overlay
media to add 4ml to each plate.
Per 50 ml Aliquot of Overlay Mix.
15ml of molten 3% LGP agarose (ONLY USE SEA PLAQUE made up
in ultra pure water and autoclaved)
35 ml of D-MEM +2% FCS ?
400 ml of sterile 1M Mg Cl
this must be kept at 39 ?C to keep the agarose molten
Add 4ml to each plate and leave in the hood to set the
agarose has a gelling temp of 26-30 ?C so it will not gel in
the incubator.
leave for 3 days and feed with 2ml of the above overlay mix.
after 6 or 7 days or if plaques can be seen fix the cells
with 10% Formal saline overnight in a sealed container.
Remove agarose overlay and add crystal violet to stain for 1hr
wash stain away under a slow tap and allow to dry.
Plaques will be Round clear spots if they are not obvious
they are not plaques
Titre (pfu/ml ) = Number of plaques per dish X 10 X
dilution.
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