Problem	Cause	Solution
Low or no signal	Cell density is too low	Increase seeding density
	Test compound or media inhibit LDH activity	Assess test compound in a system with established LDH release, by quantifying both LDHr and LDHt
	Media may contain sodium pyruvate, a known inhibitor of LDH	Avoid media with sodium pyruvate
	The incubation time for the assay may be too short	Perform a kinetic run and monitor LDH release over time (cf. (Kaja et al., 2015c)
Background signal too high / saturated	Cell concentration is too high	Optimize seeding densities
	Intrinsic LDH in serum/media is high	Reduce serum concentration in media and/or reduce MPMS Supplement concentration to 1:20,000 dilution. Heat inactivation may affect the serum LDH activity. We recommend testing the endogenous activity of serum by performing cell-free serum or complete media controls. Complete media LDH activity is typically similar to the observed LDH activity of untreated cells.
Target signal too high / saturated	Cell concentration is too high	Optimize seeding densities
	Incubation time is too long	Reduce incubation time to 30 min
	MPMS Supplement concentration is too high	Reduce MPMS Supplement concentration to 1:20,000 dilution
Dye turns yellow after addition of Stop Solution	Concentration of acetic acid is too high. The reduced dye (purple) can spontaneously oxidize back to the initial reactant (light yellow) at pH < 4.0	Reduce the amount of Stop Solution