ELISPOT Protocol
Dilute Low - Endotoxin / Azide - Free sterile unlabeled capture antibody ( BioLegend’sLEAF™ format antibodies are specifically designed for this assay ) to a final concentration of 0.5–4 μg / ml in sterile Coating Buffer and transfer 100μl / well to a high affinity binding PVDF membrane ELISPOT plate ( e . g . , Millipore ; Cat . No . MAIPS - 4510 ) .
Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours inhumidified atmosphere .
Wash plate 3 times with sterile PBS , 200 μl / well .
Add 200 μl / well of sterile Blocking Buffer .
Seal plate and incubate at room temperature for ≥ 1 hour .
Wash plate 3 times with sterile PBS , 200 μl / well .
Add appropriate sterile antigen or mitogen solution diluted in appropriate sterile tissue culture medium ( TC ) to ELISPOT plate , 100 μl / well .
Add cells diluted in sterile TC medium , 100 μl / well .
Use 50,000 - 500,000 cells / well ( the minimum number of cells should be determined in preliminary experiments ) .
Seal plate and incubate at 37°C 5 % CO2 in humidified atmosphere for theoptimum stimulation period .
BioLegend recommends a 24 hour incubation for IFN - γ , IL - 2 , and TNF - α , and a 48 hour incubation period for IL - 4 , IL - 5 , and IL - 10 for most activation conditions .
Wash plate 3 times with PBS , 200 μl / well .
Wash plate 3 times with PBS - Tween , 200 μl / well .
Add 100 μl / well of diluted biotinylated detection antibody at 0.25 - 2 μg / ml in PBS - Tween - BSA .
Seal the plate and incubate at 4°C overnight , or 2 hr at room temperature .
Wash plate 4 times with PBS - Tween , 200 μl / well .
Add 100 μl per well of the Av - HRP conjugate ( Cat . No . 405103 ) or otherenzyme conjugate diluted to its pre - determined optimal concentration in PBS - Tween - BSA ( usually between 1 / 500 – 1 / 2000 ) .
Seal the plate and incubate at room temperature for 1 – 2 hours .
Wash plate 3 times with PBS - Tween , 200 μl / well .
Wash plate 3 times with PBS , 200 μl / well .
Add 200 μl / well of fresh Substrate solution .
Monitor spot / color development at room temperature and stop reaction by rinsing plate with tap water and vigorously flicking plate over a waste container or sink , followed by blotting on paper towels or other absorbent materials .
Air dry plate overnight , until it is completely dry .
Count spots manually with a dissecting microscope or using an automated image acquisition / analysis unit ( plates can be analyzed for up to 3 months ) .
