Materials and Reagents

1. COS-7 cells (ATCC, catalog number: CRL-1651 ^TM)
2. A mammalian expression plasmid encoding a membrane protein of interest
   
3. A plasmid encoding an extracellular protein fused to the Fc portion of the human IgG [for example, pFUSE series by InvivoGen and pSXFc in Eshed et al. (2005) are options for cloning vectors to make this fusion construct]
4. Dulbecco's modified Eagle Medium (DMEM), high glucose, no glutamine (Life Technologies, catalog number: 11960-044 )
   
5. FetalClone III serum (Thermo Fisher Scientific, catalog number: SH30109.03 )
   
6. 100x GlutaMAX-I (Life Technologies, catalog number: 35050-061 )
   
7. Lipofectamine 2000 (Life Technologies, catalog number: 11668-019 )
   
8. Dulbecco's phosphate-buffered saline (DPBS) (no calcium, no magnesium) (Life Technologies, catalog number: 14190-144 )
   
9. Virus production serum-free medium (VP-SFM) (Life Technologies, catalog number: 11681-020 )
   
10. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034 )
    
11. Protein Concentrators (9K MWCO, 7 ml) (Thermo Fisher Scientific, catalog number: 87748 )
    
12. Goat anti-human IgG, Fcγ fragment specific, conjugated with a fluorophore (e.g. Jackson ImmunoResearch Laboratories, catalog number: 109-545-098 for Fluorescein and 109-585-098 for Texas Red)
    
13. COS-7 culture medium (see Recipes)
    
14. 0.2 M PB (see Recipes)
    
15. 4% PFA (see Recipes)
    
16. PBS (see Recipes)
    

Equipment

1. 6-well cell culture plates (Corning Incorporated, catalog number: 3516 )
   
2. Glass coverslips (VWR International, catalog number: 48366-227 )
   
3. 10-cm dish
   
4. Centrifuge
   
5. 37 °C/5% CO₂ cell culture incubator
   
6. A regular fluorescence microscope
   

Procedure