Please find the following protocol:


Recombinant Virus Plaque Assay


Overview:

As for any protocol involving tissue culture, employ sterile techniques throughout. Also, make sure to disinfect any virus-containing objects with bleach and/or by autoclaving.


Procedure:


Twenty-Four Hours Prior to the Assay

1. Coat 6-well cluster dishes with Poly-L-Lysine, wash with sterile PBS, and set aside. Cells will adhere better to the well when pretreated with Poly-L-Lysine.

2. Add enough 1X Trypsin-EDTA solution to cover the cells and incubate for 5 minutes.

3. Gently knock the sides of the cell culture flask to dislodge cells from the sides and immediately add a few mls of DMEM.

4. Mix the solution gently to collect the cells into the DMEM solution.

5. Plate cells in the 6-well cluster dishes at approximately 5 X 105 cells per well. Cells should be approximately 80% confluent the following day.



Plaque Assay

1. Using sterile 5 ml "snap-cap" tubes, dilute the virus containing media in DMEM with FCS as follows:

a) 1 X 103 dilution = 3 μl of virus supernatant and 3 ml of
DMEM with FCS

b) 1 X 104 dilution = 300 μl of solution a) and 2.7 ml of
DMEM with FCS

c) 1 X 105 dilution = 300 μl of solution b) and 2.7 ml of
DMEM with FCS

d) 1 X 106 dilution = 300 μl of solution c) and 2.7 ml of
DMEM with FCS

e) 1 X 107 dilution = 300 μl of solution d) and 2.7 ml of
DMEM with FCS

f) 1 X 108 dilution = 300 μl of solution e) and 2.7 ml of
DMEM with FCS

2. Discard media from the 6-well cluster dishes.

3. Add 2 ml of the above dilutions (1 x 103 to 1 X 108) to the cluster dishes and incubate for 2 to 3 hours.

4. Prepare melted 5% Agarose and keep in liquid state by heating at 55°C.

5. Dilute the 5% Agarose ten-fold with DMEM media (final Agarose concentration is 0.5%). Pipette up and down several times to ensure a homogeneous solution.

6. Dispense 3 ml per well and allow the Agarose to solidify for 10 min with the lid off in a tissue culture hood.

7. Incubate an additional 10 min with the lid on in a tissue culture hood.

8. Return cells to an incubator and incubate for between 3 to 5 days.

9. Count or pick individual plaques that appear as clear areas surrounded by a zone of rounded cells.

10. If the plaques are for counting only, cells can be stained with vital dye Neutral Red (which stains ONLY live cells). Apply 1 ml per well of Neutral Red and incubate for 1 hour. Remove the solution and continue incubation until the cells are stained a deep red.

Solutions:

DMEM with FCS.......................................10% Fetal Calf Serum
.......................................Store at 4°C in DMEM
DMEM..............................................1X Penicillin/Streptomycin
.....................................................Store at 4°C in DMEM
............................................10% (v/v) Fetal Calf Serum
Trypsin-EDTA (1X).....................1:10 of 10X Trypsin-EDTA:PBS
PBS.............................................................................pH 7.2
.......................................................................2.7 mM KCl
.....................4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
....................................................................Filter sterilize
...............1.8 mM Potassium Phosphate Monobasic (KH2PO4)
....................................................................137 mM NaCl
Neutral Red............................................1:15 Neutral Red:PBS
5% Agarose................................5% (w/v) Seqplaque Agarose
..........................................Sterilize by autoclaving


BioReagents and Chemicals

Trypsin-EDTA 10X
Neutral Red
Poly-L-Lysine
Penicillin-Streptomycin (100X)
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Agarose
Fetal Calf Serum (FCS)
DMEM


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1974