Vaccinia virus protocol

  1. Prepare TK143 cells - 3-4 large flasks of confluent cells, growing in DMEM (with glutamine) + 10% FCS + Pen/strep.
  2. Take 107 pfu aliquot of recombinant vaccinia and dilute in 5ml sterile PBS with 0.1% BSA. Tip the medium off the flasks and replace with vaccinia solution. Leave in incubator at 37C for 1.5-2 hours, agitating the flask from time to time. Make up with 25ml warm medium.
  3. Incubate cells with vaccinia for 24-48 hours. The cells should be just beginning to detach when the virus is harvested.
  4. Harvest the cells (shake the flask if necessary) and spin down, 2000rpm for 10 minutes.
  5. Tip the supernatant into Virkon and resuspend the pellet in 3mls of PBS/1% BSA. Freeze and thaw rapidly 3 times using a waterbath at no more than 37C and a dry ice/alcohol bath. Vortex thoroughly after each thawing. 50ml Falcon tubes are best for this process. After the last freeze/thaw cycle, sonicate the mixture thoroughly.
  6. Spin down hard and transfer the supernatant to 1ml freezing vials.
  7. Titre the virus on TK cells in 6 well plates. These are prepared by resuspending TK cells at 1.25 or 2x105/ml and adding 2.5ml to each well, then leaving for 24 hours to become confluent. Make dilutions of the harvested vaccinia stock to 106,107 108. Remove the medium from each well and replace with 0.5ml of virus, in duplicate. (Need 1ml of each dilution). Leave for 30mins to 1 hour then add 2ml of warm GM and incubate for 48 hours.
  8. After 48 hours, remove the medium and fix the cells with 2ml of 4% formalin in PBS for 20 minutes at room temperature. Remove the formalin, then stain with 1ml of Giemsa stain (1/8 dilution).
  9. Count the number of clear plaques in the two wells and multiply by the dilution factor to give the titre.

(e.g. 6 plaques in 2 wells at 10-8 equals 6x108 pfu/ml).

To make the dilution series:

Volume (ml)

of What

to add to (ml) of PBS/0.1% BSA

Resulting

Dilution

5

Vaccinia

495

10-2

5

10-2

495

10-4

15

10-4

1485

10-6

150

10-6

1350

10-7

150

10-7

1350

10-8

 

To make titration plates:

For 1 plate: 2x106 cells into 15ml GM (DMEM + 10%FCS/1%P+S/1%Glutamine)

For 3 plates: 6 x106 cells into 48ml GM

For 6 plates: 12 x106 cells into 96ml GM

For 9 plates: 15 x106 cells into 144ml GM

For 16 plates: 32 x106 cells into 256ml GM