ELISpot Troubleshooting

1. Make sure not to touch the bottom of the ELISpot plate wells, avoiding puncturing the membrance with pipet tips.

2. Optimize the cell concentrations (eg, 103 – 106 cells per microwell) in the first experiment.

3. Do not disturb the incubator or ELISpot plate during the cell culture process to avoid streaks and ambiguous spots.

4. High background can most likely be overcome by performing the following steps:

• Soak and wash the plate with PBS (without Tween) prior to adding substrate.

• Enough times and intensity of washing with PBS-Tween.

• Sufficiently dry the plate.

• Prevent the carryover of natural cytokines and wash cells thoroughly prior to the experiment.

• Do not overdevelop.

5. Do not dry the plate at a temperature higher than 37°C to avoid cracking of the membrane filters.

6. Store color-developed, dried plates in darkness to avoid color attenuation.




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