and Reagents for Selecting Phoenix Cells
the Phoenix cells with both of the following at the same time:
B from boerhinger mannheim; cat.# 843-555, 1g/50mls; you want 200ug/ml
as the final con. so use this as 250X stock solution. select for
toxin from calbiochem; cat# 322326, 1 gram. therefore dissolve in
sterile PBS with 0.1% BSA, aliquot and store at -80íC. use at 2ug/ml
on cells. select for 1 week as well.(dissolve in 1 ml. which will
give you 500 stock solution)
one week of selection and another week of regrowth, your cells will
produce high titre virus.
- You only
need to put the cells under drug selection once every 3 months. However
this is not really always necessary as the cells are pretty much stable!
Only if you see a significant drop in titre should you put the back
into drug selection. and always check titre with standard retroviral
vectors containing either LacZ(pBabeMNLacZ) or GFP(pBabeMN-IRES-GFP).
- For detecting
the Env surface protein, use supernatant from hybridoma 83A25.
Envelope antibody from hybridoma 83A25. Must obtain Material transfer
Agreement from Dr. Frank Malick, Lab of Persistent Viral Diseases, National
Inst. of Allergy and Infectious Disease, Hamilton, Montana. Phone: (406)
363-9374. Or, call
- Dr. George
Keller at the Office of Tech. Licensing(OTL) at NIH; (301) 496-7735
ask for Patent no. USPN 4,405,712 Hybridoma 83A25.Ref. Evans, 1990
"A neutralizable epitope common to the envelope glycoproteins of
ecotropic, polytropic, xenotropic, and amphotropic murine leukemia
Virol 64, 6176-83 (1990) 
antibody for anti Env = Goat anti Rat IgG/P.E. conjugate from Caltag,
- For detecting
gagpol, we check for the surface marker Lyt2(mouse CD8) by using
53-6.7; Rat IgG2A Kappa; We use a FITC conjugate: Cat.# 01044D