Purification of VSV or SeV

Sample(s):

1. If harvested supernatant was frozen, thaw in 37C waterbath.

2. For each sample type, split supernatants among (12 if from 20 T75; process in two batches) Beckman 1 x 3.5in. thick wall polycarbonate centrifuge tubes (Up to 35ml per tube). Underlay supernatant with 5ml 20% (w/v) sucrose in HEN buffer using a serological pipette.

3. Centrifuge at 28k rpm and 4C for 3.5hr in a SW32 Ti rotor using the Beckman Coulter
Optima L90k Centrifuge. Decelerate using “slow” setting.

4. Following centrifugation, discard supernatants and invert tubes over paper towel with plastic
wrap underneath to dry. If necessary, use a cotton swab to remove the last of the liquid from
around the rim.

5. Add 80ul of HEPES buffer saline (HBS) to each tube, cover with parafilm and
incubate at 4C overnight to resuspend pellet.

6. Prepare(1 per every 2 1x3.5in tubes) 11ml 7.5-27.5% continuous iodixanol (diluted in HBS) gradients in Beckman
9/16 x 3.75 in. UltraClear Centrifuge Tubes

a. Place 1.2ml of 27.5% iodixanol in the bottom of the tube and successively overlay with 1.2ml of 25%, 22.5%, 20%, 17.5%, 15%, 12.5% and 10% iodixanol. Finish with 1.4ml 7.5% iodixanol.

b. Cover with parafilm and incubate overnight ( hrs) at 4C to linearize gradient.

7. Layer resuspended virus on top of the iodixanol gradients.

8. Balance with HBS and centrifuge at 26.5k rpm and 4C for 30min using a SW40 rotorand
Beckman Coulter Optima L90k Centrifuge.

9. 10. Remove viral band from gradient

a. Mark the viral band with a fine tipped Sharpie.

b. Use a 5ml pipette to carefully remove all the material above the viral band.

c. Use a new 5ml pipette to remove all of the viral band

d. Transfer this material to (1 per every 3 gradients) new Beckman 9/16 x 3.75 in. UltraClear Centrifuge Tubes.

10. Add 10ml ETto each tube, cover with parafilm and mix by inversion.

11. Balance with ET and centrifuge at 27k rpm and 4C for 1.5hr using a SW40 rotor and
Beckman Coulter Optima L90k Centrifuge.

12. Following centrifugation, discard supernatants and invert tubes over paper towel with plastic
wrap underneath to dry. If necessary, use a cotton swab to remove the last of the liquid from
around the rim.

13. Resuspend pellets in ET buffer with 10% DMSO.(Typically around 500ul per tube)

-Note: If virus is intended for animal studies can also resuspend in PBS. However, virus will be less stable.

14. Store at -80C in “ b”.

Current as of 12/10/14

HEPES Buffer Saline (HBS) .

21mM HEPESFor 1L:

140mM NaCl5.0g HEPES free acid

45mM KCl8.2g NaCl

0.75mM Na2HPO43.4g KCl

0.1% (w/v) Dextrose0.106g Na2HPO4

pH7.5 (with NaOH)1g Dextrose

Filter sterilizeddH20 to 1L

[Based on Swenson et al. Clin. Vaccine Immunol. 2008]

Iodixanol % / Ratio 60% Optiprep:HBS / 60% Optiprep for 9.6ml (ml) / 1x HBS for 9.6ml (ml)
27.5 / 11:13 / 4.4 / 5.2
25 / 5:7 / 4.0 / 5.6
22.5 / 3:5 / 3.6 / 6.0
20 / 1:2 / 3.2 / 6.4
17.5 / 7:17 / 2.8 / 6.8
15 / 1:3 / 2.4 / 7.2
12.5 / 5:19 / 2.0 / 7.6
10 / 1:5 / 1.6 / 8.0
7.5 / 1:7 / NA / NA

For 11.2ml 7.5% iodixanol mix 1.4ml 60% Optiprep with 9.8ml of 1x HBS

Current as of 12/10/14