Materials:
30
ml of exponential culture of SF9 cells at 5x105 cells/ml
6-well
plates (2 each)
1
bottle 4% Agarose Gel
1
bottle SF-900 (1.3X) medium
1
sterile bottle
0.5
ml baculovirus supernatant
100
ml SFM
1.
Under
sterile conditions dispense 2 ml of cell suspension per well.
2.
Allow
cells to settle to bottom of plate and incubate, covered, at RT for 1h.
3.
Place
the bottle of agarose gel in the 70oC water bath. Place the empty
bottle and the SF-900 (1.3X) in the 40oC bath.
4.
Following
1h incubation of the plates at RT, observe monolayers under the inverted
microscope to confirm cell attachment and 50% confluence.
5.
Produce
an eight-log serial dilution of the harvested viral supernatant by sequentially
diluting 0.5 ml of the previous dilution in 4.5 ml of SFM medium in 12-ml
disposable. You should conclude with 8 tubes containing each of a 10-1
to 10-8 dilution of the original virus stock.
6.
Move
the six well plates and the tubes of diluted virus to the hood. Label the
plates, in columns of two, “10-3, 10-4, 10-5,
10-6, 10-7, 10-8”.
7.
Sequentially
remove the supernatant from each well, discard, and immediately replace with 1
ml of the respective virus dilution to each duplicate well. Incubate for 1h at
RT.
8.
Move
bottles from waterbaths to sterile hood when agarose has liquid (20 to 30 min).
Quickly dispense 30 ml of the SF-900 (1.3X) medium and 10 ml of the 4% agarose
gel to the empty bottle and mix gently. Return the bottle of plaquing overlay
to the 40oC water bath until use.
9.
Following
this second 1h incubation, return the bottle of diluted agarose and the 6-well
plates to the hood.
10.
Sequentially
(from high to low dilution) remove the virus from the wells and replace with 2
ml of the diluted agarose. Work quickly to avoid desiccation of the monolayer.
A Pasteur pipet connected to a vacuum pump easily removes inoculum traces.
11.
Allow
gel to harden for 10 to 20 min before moving.
12.
Incubate
at 27oC in a humidified incubator for 4 to 10 days.
13.
Recombinant
virus produces milky/gray plaques of slight contrast visible without staining
or other detection methods.
14.
Monitor
plates daily until the number of plaques counted does not change for two
consecutive days.
15.
To
determine the titer of the inoculum employed, an optimal range to count is 3 to
20 plaques per well of a 6-well plate. The titer (pfu/ml) may be calculated by
the following formula:
16. Overlay of agarose with Natural Red dye:
Prepare 0.5% agarose
solution in SFM
Add Natural Red solution to
a final concentration of 50mg/ml (dilute 3.33mg/ml stock 1:66.6).
Overlay 1ml of the dye
solution to each well (on a 6 well plate).
Pfu/ml (of original stock) = 1/dilution factor x number of plaques x
1/(ml of inoculum/plate)