Adenovirus Amplification & Purification
JK Lab 2006
# AD293 cell Media :
10% FBS + 1% PS in High Glucose w/ 4mM Glutamine DMEM
# Spin down always should be done @4°C.
Initial Expansion Step
1. Prepare one Ti-25 flask with AD293 cells at least 24hrs before transfection
(cells should be confluent more than 70% when you transfect)
2. Transfect 5ug of Viral plasmid in 2.5ml of OPTI-MEM
(5~10ug Viral DNA + 20ul Lipofectamine + 500ul OMTI-MEM followed by
our lab transfection protocol. Modify viral plasmid amount if need)
3. After 6 hrs, remove the media & add 5ml fresh media
4. Next morning, remove the media again & add 5ml fresh media.
5. Keep growing and check the cells every day. Every 3 days, change media
carefully, but when cells start to float, just add 2ml fresh media. Generally,
CPE shows up after 1 week.
6. When you can see >70% of cells are floating, harvest cells in 15ml falcon
tube.
♠ Spindown @1000rpm for 5min. (Do not spin @high speed. Otherwise,
cells would be broken).
♠ After spindown, wash with 3ml of DPBS once. Spin again and get rid of
all supernatant.
♠ Resuspend cell pellet with 2~3ml of DPBS in 15ml Falcon Tube.
♠ Go to freezing/thawing with methanol/dry ice bath
(Ethanol/dry ice bath is also fine).
♠ Freeze in Ethanol/dry ice bath for 5min and thaw in 37°C water bath for
2 min. (Do not keep in 37°C water bath more than 5min)
♠ Vortex vigorously and repeat two more cycles (Total 3 cycles).
♠ After freezing/thawing, spindown @3000rpm for 10min, and take
supernatant.
♠ Save 50% of your supernatant for backup and keep @-80°C.
 Finally your viral solution volume is around 2~3ml
Amplification Step
1. Prepare 5 X 15cm Plate of AD293 cells at least 24hrs before infection
(When you infect, cells should be more than 70% confluent)
2. Before you infect, mix 1~2ml of your 1st viral supernatant with 75ml of
AD293 cell media in Ti-75 flask. Mix very well and add 15ml to each plate.
 Don’t use more than 15ml media. Otherwise, infection efficiency would
be low. It will take longer time to infect.
3. Generally, it takes 4~5 days to see full CPE effect.
♠ Around 2nd ~3rd day, you can see AD293 cells start to change their
morphology. They going to be round up and start to be detached from the
plate. Check every morning and night.
♠ Wait until you can see full CPE effect until every cell is ready to float.
♠ When you can see every cells are shrink and some cells are floating,
harvest all.
♠ Divide into 2 X 50ml Falcon tubes and spin down @1000rpm for 3min.
♠ Get rid of supernatant and wash the pellet with 5ml DPBS/tube once.
♠ Spin again, get rid of supernatant, and combine all cell pellets with 6 ml
of DPBS.
♠For easy freezing/thawing, you had better divide your 6ml DPBS
cell pellet into 2 X 15ml falcon tube (each tube will contain 3ml).
Small volume is much easy for freezing/thawing.
♠ Go to Freezing/thawing for total 3 times and spindown @3000rpm for
10 min.
♠ Take only supernatant. Save 50% for your backup. Keep @-80°C.
 your viral volume would be around 6ml. If you want, save 50% @-80C
Large Scale Expansion & Purification Step
1. Prepare 20 X 15cm Plates of AD293 cells before infection. Wait until cells
are >90% confluent.
2. Mix 3ml of your previous viral supernatant with 300ml of AD293 cell media
in Ti-150 flask. Mix very well and add 15ml to each plate.
3. Generally, you can see full CPE within 24~48hrs. In my case, I can see full
CPE effect 24hrs after infection.
♠ Harvest all and divide into 6 X 50ml falcon tube.
♠ Spindown @1000rpm for 5min. Remove supernatant and wash w/ 5ml
DPBS.
♠ After wash, spin again and combine all cell pellets with 8ml of DPBS.
♠ Divide your cell lysate in 2 X 15ml falcon tube (4ml each)
♠ Go to Freezing/Thawing for total 3 times.
♠ After freezing/thawing, spindown @3000rpm for 10min and get
supernatant.
♠ Go to CsCl2 Purification or keep @-80°C
4. Prepare 1.25g/ml CsCl2 solution & 1.40g/ml CsCl2 solution.
Autoclave. Store @RT
♠ 1.25g/ml CsCl2 solution : 36.1g CsCl2 in 100ml of 10mM Tris pH 8.0
♠ 1.40g/ml CsCl2 solution : 64.0g CsCl2 in 100ml of 10mM Tris pH 8.0
5. Prepare 2L of 10% glycerol/PBS. Autoclave and store @4°C (Should be
chilled before dialysis)
6. Assemble 2 X CsCl2 gradient tubes.
♠ Add 3.5ml of the 1.25g/ml CsCl2 solution into each SW41 Centrifuge
tube (Beckman Polyallomer centrifuge tube, 14 X 89mm). No bubble !!!
♠ Using the smallest pipette, aspirate 3.5ml of 1.4g/ml CsCl2 solution,
place the pipette tip at the bottom of the tube, and slowly dispense
solution under the 1.25g/ml solution. No bubble !!!
♠ Aspirate 4ml of the viral solution. Place the pipette tip against the side
of the tube above the CsCl2 solution. Gently release the cell lysate on top
of the gradient.
♠ Add 300~500ul mineral oil. Check balance w/ mineral oil.
7. Centrifuge in the ultracentrifuge @ 35000rpm for 18 hrs, 4°C.
(optional : Additional CsCl2 gradient can be used. 1.35g/ml)
8. Using 18½ gauge needle & 3ml syringe, pull out the viral band from 2 X
CsCl2 gradient tube. Total volume would be around 3ml.
9. Dialysis with pre-chilled 1L of 10% glycerol / PBS in cold room. Use
10000 MWCO dialysis cassettes (Pierce, #66425). After 1 hour, change
liquid. Do dialysis one more hour.
10. After dialysis, take virus solution and aliquot 200ul, keep @-80°C
11. Measure the total viral amount
1 unit of A260 = 1.0 X 1012 viral particles / ml
Generally, you may have 1 active viral particle among 100 viral particles