Protocols - Selection

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 -80’C. 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.