Materials and Reagents

1. Salmon sperm DNA (Life Technologies, Invitrogen™, catalog number: AM9680 )
   
2. Dulbecco’s modification eagle medium (DMEM) (Life Technologies, Invitrogen™)
   
3. Ammonium chloride (0.17 M, filtered, autoclaved) (pH 7.4)
   
4. 10% DMEM: 10% FBS in DMEM
   
5. Biotin conjugated anti-IgM or anti-IgG (Southern Biotech)
   
6. Streptavidin alkaline phosphatase (Southern Biotech)
   
7. 5-Bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich)
   
8. 10x PBS-Tween 20 (see Recipes)
   
9. Blocking solution (see Recipes)
   
10. AMP buffer (see Recipes)
    

Equipment

1. 45-μm syringe top filter (USA Scientific)
   
2. Cell-culture incubator
   
3. Clear 96-well Microtest polystyrene assay plate (BD Biosciences)
   
4. Conical tubes (Corning)
   
5. Dissecting microscope or an ELISpot reader
   

Procedure