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|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.|