Table 2. Troubleshooting for in vivo experiments.

Very low or non-existent signal	Low luciferase expression in the selected animal model	Overall, light emission from protease-specific bioluminescent probe is expected to have a lower intensity that light resulted from D- luciferin injection. If light emission generated by D-Luciferin is already low, increase dosage of protease- specific peptide and CBT
	Quality of reagent issue	Verify that the peptide probe is not oxidized or degraded using HPLC/MS analysis
Low signal to background ratio (no difference in light emission between treatment and control groups).	Low level of protease activation	Increase drug/effector used to activate (or decrease) protease activity
		Verify protease activation using commercial probe (if available) or use classical ex vivo methods
	Quality of protease-specific peptide is compromised	Verify that the peptide probe is pure and not oxidized or degraded. HPLC/MS can be used for analysis
	Low purity of peptide	Very high degree of purity is necessary as traces of Free D- Cysteine can result in unspecific formation of D-luciferin that significant contribute to background. Verify peptide purity using HPLC and MS analysis. If stored solution was used, prepare a fresh one