Author
Date
Version
SOP #
Charles Lefebvre
May 2011
1
V005
Objective: This SOP addresses the titration of VSV or Maraba virus by plaque assay on
VERO cells
Required reageants/equipment:
-DMEM + 10% FBS
-1% agarose (made up in dH2O)
-2 x DMEM + 20% FBS: GIBCO #12100-046, dissolve powder (13.4g) in less then 400ml,
add 3.7g sodium bicarbonate. Top up to 400 ml, 0.22um filter, add 100 ml FBS.
-DMEM
-Methanol:Acetic Acid solution (3:1 Methanol:Acetic Acid)
-0.2% crystal violet (in 20% ethanol) – made up from stock 5% crystal violet in 100%
methanol
SOP-V005: Plaque Assay of VSV or Maraba virus
Day One – Seeding Cells
1. Seed VERO in 2 (6 well plate) at 6^105 cells / well using DMEM + 10% FBS.
Day Two – Plaque Assay
2. Warm up complete media, 1% Agarose (to solubilize ~ 200ml of agarose put in
microwave for 2.5 mins.) and 2X DMEM + 20% FBS at 48°C.
3. Prepare virus dilutions in media (no serum). Need 100 ml per dish or well. Plaque
assay is done in duplicate. Remember a “negative control” plate with 100 ml media
alone.
(i) For dilutions prepared for round 2 media stock (from xE6 to xE10) + UIF
or
(ii) For dilutions prepared from sucrose cushion purified virus (from xE8 to xE12) +
UIF
Do dilution done in 24 well plate:
10ul
100ul
100ul 100ul
cold dMEM: 990ul 900ul
(no serum)
dilution:
xE2
900ul
100ul
900ul
Final dilution: xE3 xE4
xE5
xE6
On plate
(Because you add 100 ul from 1000ul)
100ul
100ul
100ul
900ul
900ul
900ul
xE7
xE8
xE9
900ul
xE10
This protocol is shared by the Stojdl Lab under a creative commons license.
(iii) For dilutions prepared from a virus plug (in 500 ul PBS) (from xE2 to xE6 + UIF)
100ul
100ul
100ul 100ul
cold dMEM: 900ul 900ul
(no serum)
dilution:
xE1
900ul
900ul
Final dilution: xE2 xE3
xE4
xE5
On plate
(Because You add 100 ul From 1000ul)
4. Aspirate to remove medium from VERO cells.
5. Add 100 ml diluted virus per plate to cells dropwise. Can do 5 dishes at a time but
do not let cells dry out.
6. Add 200 ul of warm complete media
7. Incubate 37oC for 60 minutes, rocking every 15 minutes or so to allow for
attachment.
8. Mix enough agarose overlay (2ml/well) (1:1 1% agarose: 2x media + 20% FBS)
9. Add 2 ml/ (well), dropwise of agarose overlay (1:1 1% agarose: 2x media + 20%
FBS). Do NOT swirl after addition of agarose to dish(well). Let solidify for 10 min in
hood. Incubate o/n at 37oC.
Day Three – Counting Plaques
10. Plaques are visible and can be counted by eye. You also have the option of verifying
your counts by staining cells to facilitate visualization.
11. Staining plaques (optional): Fix cells through agarose overlay by adding 2
ml/dish(well) of Methanol:Acetic Acid solution (3:1 Methanol:Acetic Acid). Let sit 30
minutes at room temperature.
12. Under gently flowing warm tap water, carefully “rinse” out agarose layer. Direct
stream of water to side of dish(well) and should gently slid agarose layer off.
Discard agarose layer.
13. Add minimal amount of 0.2% Crystal Violet to each dish(well), just enough to cover
bottom. Let sit for roughly 30 minutes on orbital shaker.
14. Gently rinse each dish(well) with water. Tap off excess water. Allow monolayer to
dry. Count plaques.
Note: instead of crystal violet you can use coomassie blue staining solution:
coomassie blue (0.4g)
120 ml H2O
80 ml methanol
Stir 5 min
filter 0.2 uM
40 ml acetic acid
160 ml H20
This protocol is shared by the Stojdl Lab under a creative commons license.