Neutralization of BPV1-Induced Foci in C127 Cells

Diana Pastrana and Chris Buck

John Schiller’s Group

Laboratory of Cellular Oncology, NCI

Use C127 clone C (originally cloned by Bill Vass) (second box down in “Ralph” rack on far right-hand side of liquid nitrogen tank)

Medium:  DMEM, 10% HI FCS, 1x Glutamax-I, 50 µg/ml Primocin.  Sterile filter the completed medium.

Thaw cells (1.5 million per aliquot) in 10 ml of medium.  Spin down, plate in 5ml of medium in a 25 cm²flask.  The cells may take several days to recover. 

One T-75 flask yields enough cells for 10 x 60mm dishes (4 x 6-well plates)

Pre-plate cells at 1.5 x 10⁶cells per 60 mm plate or 0.6 x 10⁶cells per 6-well well.  

For 6-well plates (remainder of protocol) use 3ml per well (0.2 x 10⁶cells per ml).

The day of infection, remove 2 ml from each well (leaving 1 ml).  If reagents are highly limiting, 0.5 ml is adequate to keep cells covered. 

Add neutralizing agent to cells.

Add 0.05 µl of Patricia’s BPV1 virion stock to each well.  Return cells to the incubator.  Gently rock the plates every 15 minutes or so for the first hour.

Incubate overnight.

Remove virus inoculum. Add 3 ml of fresh medium.

Replace medium every Tuesday and Friday for 17 days or until foci are large enough to stain.  The first microscopic hints of foci (rounded cells above the plane of the monolayer) come at about 7-10 days.

Stain foci.  Remove medium and add ~1ml of stain stock (12.5g methylene blue + 2.5g carbol fuchsin in 500ml methanol – store indefinitely) to each well.  Put on rocker for several minutes (room temp).  Rinse the wells gently with diH₂O.  Gently agitate plate in a bucket with ~1 liter of warm water + ~50ml of RocalD. Pour RocalD solution out of plate and repeat until background is appropriately low.  A final gentle rinse with diH₂O may be helpful.