For further assistance, please contact our technical service department.
| 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. |