Problem/issue	Possible causes	Remedy/solution
High background/Poorly-defined or confluent spots	High numbers of dead cells.	Assess cell viability before culture set-up and stimulation.
	Inadequate membrane pre-wetting steps.	Make sure to respect pre-wetting time and that membrane turn to gray after 1 min. 35% ethanol should be prepared immediately before use.
	Plates moved during the incubation period.	Do not move plates to avoid poorly-defined snail trail spots.
	Wash steps.	Manual wash is more efficient than plate washers.
	Formation of protein aggregates.	Filter both capture and detection antibodies to reduce background and false positive spots.
	Secondary and detection antibody concentration.	Adjust biotinylated secondary/detection antibody concentration to reduce background.
	Cold developing reagents.	Bring substrate to room temperature to avoid underdevelopment.
No spots/Blank wells	Poorly defined spots.	Do not stack plates during incubation period to have a homogeneous temperature in the different wells.
	Stimulation substrate not well prepared.	Bring the peptide mixture aliquot to room temperature for 15 min to avoid crystal formation.
	Cell clumping.	Resuspend cells into single-cell suspension to avoid underestimation of spot-forming units.
	Cells not stimulated appropriately.	Use a polyclonal activator as a positive control.
	Inhibition of enzyme reaction by tween-20.	Wash plates with PBS before adding BCIP/NBT substrate solution.
	Wrong antibody pairs.	Make sure that the capture and detection antibodies react with different antigenic epitopes.