Materials and Reagents

1. 293T cells (ATCC)
   
2. pAdVAntage (Promega Corporation, catalog number: E1711 )
   
3. pCMV4-BlaM-Vpr (Addgene, catalog number: 21950 )
   
4. pNL4-3 proviral DNA (NIH AIDS Reagent Program) or TN6-GFP encoding primary Env (available upon request to Warner Greene)
   
5. DMEM (Mediatech, Cellgro^®, catalog number: 10-013-CV )
   
6. RPMI 1640 (Mediatech, Cellgro^®, catalog number: 15-040-CV )
   
7. 1x phosphate-buffered saline (PBS)
   
8. Fetal bovine serum (FBS)
   
9. 100 U/ml of penicillin and 100 U/ml of streptomycin (Life Technologies, Gibco^® catalog number: 15140-122 )
   
10. 2 M CaCl₂
    
11. Alliance HIV-I p24 ELISA kit (PerkinElmer, catalog number: NEK050B001KT ) or FlaQ assay reagents (Hayden et al., 2003)
    
12. Peripheral blood lymphocytes (PBLs)
    
13. CCF2-AM substrate and loading solutions (Life Technologies, catalog number: K1032 )
    
14. CO₂-independent media (Life Technologies, Gibco^®, catalog number: 18045-088 )
    
15. Probenecid (Sigma-Aldrich, catalog number: P8761 )
    
16. Mouse antihuman CD3 conjugated to APC-Cy7 and mouse antihuman CD4 conjugated to PE-Cy7 (BD Biosciences, catalog numbers: 557832 and 557852 )
    
17. BD CompBeads (BD Biosciences, catalog number: 552843 )
    
18. 16% Paraformalehyde (Electron Microscopy Sciences, catalog number: 15710 )
    
19. HBSS (see Recipes)
    
20. Dulbecco’s modified Eagle medium (DMEM) culture media (see Recipes)
    
21. Roswell Park Memorial Institute (RPMI) culture media (see Recipes)
    
22. CCF2 loading solution (see Recipes)
    
23. Stock solution of probenecid (250 mM) prepared in 250 mM NaOH (see Recipes)
    
24. Development media (see Recipes)
    

Equipment

1. T175 cm² culture flasks
   
2. 96-well V-bottom plate (Corning Incorporated, catalog number: 3363 )
   
3. 5, 10, 25 ml pipettes
   
4. 50-ml Falcon tube
   
5. 0.22 μm poresize Steriflip (EMD Millipore, catalog number: SE1M179M6 )
   
6. 2 ml Nalgene tubes (Thermo Fisher Scientific, Nalgene^®, catalog number: 5000-0020 )
   
7. Clear ultracentrifuge tubes (BD, catalog number: BD340437 )
   
8. 37 °C, 5% CO₂ incubator set at 90% humidity
   
9. Ultracentrifugation equipment with SW28 rotor
   
10. Flow cytometer
    Note: The fusion assay alone requires a flow cytometer equipped with a violet laser excitation (405 nm) and two measurement parameters. The photomultiplicator tube (PMT) with a 450/50 nm band pass filter, commonly used for the detection of Pacific Blue, is used for the detection of the cleaved CCF2 substrate. The other PMT with a 515/20 nm band pass filter, commonly used for Amcyan detection, is used for the detection of the uncleaved CCF2 substrate. Additional PMTs are necessary for the measurement of the fluorochromes associated with the CD3 and CD4 antibodies. APC-Cy7 is excited by 633 nm red laser and detected with a PMT with a 755 long pass filters. PE-Cy7 is excited by a 531 nm yellow-green laser and detected with the PMT with a 755 long pass filter.
    

Software

1. FlowJoX software (Tree Star) or other FACS analysis software
   

Procedure