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Dept of Molecular Pharmacology
Dept of Microbiology & Immunology

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Parameters and Reagents for Selecting Phoenix Cells

Phoenix drug selection:

  • Select the Phoenix cells with both of the following at the same time:
    • Hygromycin 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 1 week.
    • Diptheria 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)
  • After 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 FACS Sorting:
  • For detecting the Env surface protein, use supernatant from hybridoma 83A25.
  • Anti Envelope antibody from hybridoma 83A25. Must obtain Material transfer Agreement from Dr. Frank Malik, Lab of Persistent Viral Diseases, National Inst. of Allergy and Infectious Disease, Hamilton, Montana. Phone: (406) 363-9375; fmalik@niaid.nih.gov OR contact Dr. Leonard Evans (406) 363-9374; levans@niaid.nih.gov
  • Secondary antibody for anti Env = Goat anti Rat IgG/P.E. conjugate from Caltag, Cat.# R40104
  • For detecting gagpol, we check for the surface marker Lyt2(mouse CD8) by using
    • Murine CD8(Lyt-2) antibody:
    • clone 53-6.7; Rat IgG2A Kappa; We use a FITC conjugate: Cat.# 01044D
    • Pharmingen.

     

 

   

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