Troubleshooting Guide: ELISpot

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Observation Problem Corrective Action
Following the incubation with BCIP/NBT chromogen and rinsing the microplate with deionized water, the dark-blue background color of filter membrane attenuates visualization and quantitation of spots. Wet membrane Microplates cannot be analyzed accurately until PVDF filter membranes are completely dry. Wait until membrane becomes dry, usually 15 - 30 minutes at 37° C or 60 - 90 minutes at room temperature
The number of spots in the wells that contained the cells is high but their contrast as well as intensity of staining in the Positive Control wells is low. Underdevelopment - may be a result of using Streptavidin-AP and/or BCIP/NBT solutions that have not been brought to room temperature Bring reagents to room temperature before adding to the wells.
The number of spots in the wells that contained cells is lower than expected whereas Positive Control wells turned black-blue. Cell stimulation problem Ensure that reagents used to stimulate the cytokine release from the cells retained their biological activity. One way to check is to perform immunocytochemistry on fixed cells after stimulation.
Too few cells added to the wells Increase the number of cells added per well.
Following incubation with BCIP/NBT and drying the microplate, the density of the spots makes it difficult to quantify them. Too many cells were added to the wells Make dilutions of cells (i.e., 1 x 106, 5 x 105, 1 x 105, 5 x 104, 1 x 104 cells per well) to determine the optimal number of cells that will result in formation of distinct spots.