**Coat the Plate:** 1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s LEAF™ format antibodies are specifically designed for this assay) to a final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100 μl/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 in humidified atmosphere. **Block the Plate:** 1. 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. Set-Up Tissue Culture and Add Antigen or Mitogen: - 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 the optimum 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. **Add Detection Antibody:** 1. 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. **Add Avidin-Horseradish Peroxidase (Av-HRP):** 1. Wash plate 4 times with PBS-Tween, 200 μl/well. - Add 100 μl per well of the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate diluted to its pre-determined optimal concentration in PBS-Tween-BSA (usually between 1/500 – 1/2000). Source: 1. postech.ac.kr 2.http://www.biolegend.com/media_assets/support_protocol/BioLegend_ELISPOT_protocol.pdf