. 2020 Jan;185:111196. doi: 10.1016/j.mad.2019.111196

Table 2.

Troubleshooting tips to use the mito-QC Counter.

Problem	Solution
Not enough dots detected.	Decrease the Ratio image threshold. (ex:0.4)
Not enough dots detected.	Decrease the spot red intensity threshold. (ex: mean(0) -1 stDev).
Too many dots detected.	Increase the Ratio image threshold (ex:0.6)
Too many dots detected.	Increase the spot red intensity threshold (ex: mean(0) +1 stDev).
Unspecific dots (especially at the border of your cell).	Increase the spot red intensity threshold (ex: mean(0) +1 stDev).
Picture is very noisy.	Increase the radius for smoothing
High background fluorescence can lead to excessive detection of mitolysosome area and unreliable counting.	Optimise experimental conditions to reduce background. We advise avoiding the use of mounting media containing nuclear dyes. Instead, counterstaining with nuclear dyes is possible if performed before the mounting step.
Heterogenous population of cells expressing the reporter (viral or transient transfection).	Estimation of mitochondrial content and linked parameters cannot be used in these conditions. If these parameters need to be assessed, we advise FACS sorting the cells to obtain a homogenous population.
Bleaching of mCherry and/or GFP signal can lead to different ratios between pictures of the same sample and aberrant results in the mitophagy mask.	Optimise sample preparation and/or acquisition parameters to reduce bleaching.