DENGUE VIRUS GROWTH IN C636 INSECT CELLS (Ana/Dabeiba)
Materials
C636 cells growing in T 75 flasks (10-12 flasks)
C636 medium (see below).
Cell incubator (28oC, 5% CO2)
Dengue virus inoculum (Dengue-2, Dengue-3, etc..)
1X PBS w/BA & P/S
Procedure
A day before of infection plate 2.0x10^6 C3B6 cells / flask .
Wash Cells with PBS 1 X . Infect cells with Dengue virus at MOI=0.01 (approx 2.0x 104
IP/flask). Adding 2mls/flask of diluted dengue virus in 1X PBS/BA/PA . Let the virus
adhere for 1 hour at Room Temperature . After the incubation period , remove the 2 ml
of virus and add 10 mls of C636 medium to each flask. Incubate for 7 days at 28oC, 5%
CO2.
To harvest virus, remove all the media from the infected flasks and pool them into 50 ml
tubes. Spin down tubes at 1000 rpm for 5 minutes and collect the supernatant. Aliquote
and quick freeze with dry ice-ETOH. Store viral aliquots at -80 oC.
C6/36 Medium Recipe
500 ml RPMI (Gibco )
50 ml Fetal Bovine serum
15 ml of NaHCO2 5% solution
5 ml Na Py
5 ml Non Essential Amino acids
5 ml L-Glutamine
1X PBS w/BA & P/S Recipe
To a sterile ½ L bottle add
50 ML 10xPBS
437 ML of ddH20
Autoclave ( liquid setting ) once is autoclaved
Add
5 ml 100X P/S
3 ml 35% BA
Mix WELL
Add last
5 ml 100X Ca/Mg
Total 500 ml
100x Ca/Mg Recipe
1.327 g CaCl2● 2H20(Calcium Chloride )
2.133 g MgCl2● 6 H20 ( Magnesium Chloride )
Add dd H2O up to 100 ml
Autoclave (Liquid settings )
C6/36 thawing out and growth.
Rapidly thaw cells from liquid Nitrogen
Add 9 ml of RPMI
Centrifuge 1200 rpm 10 min
Remove supernatant
Resuspend in 1 ml of C6/36 medium
Add cells to 4 ml of C6/36 medium in a small flask
Lay down flasks . Incubate at 28 C 5% CO2
Check every day
Ad 5 ml of C6/36 Medium according with growth