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).
