Increase the cell incubation time or use indirect method (pre-treat cells with stimulant).
Use a positive stimulation control – a stimulant that you know will induce expression of your cytokine/protein of interest.
Monitor color development with an overhead microscope and ensure that the developing reagents have been stored correctly and have not lost activity.
Increase the number of cells. Cell numbers should usually range between 1- 2 X 105 cells per well, and this may require some optimization.
Concentration of the primary and/or secondary antibody will need to be increased. This will require optimization.