Table 2.

Troubleshooting table

Step	Problem	Possible reason	Solution
2,4,5	Cell death occurs	High concentration of FreSHtracer	Test the toxicity of FreSHtracer in cells of interest and load probes in the non-toxic concentration range
		Phototoxicity	Adjust laser power or time interval for image acquisition
2-B-i	MitoFreSHtracer stains mitochondria unevenly	Low solubility of MitoFreSHtracer in medium	Add 0.03% Pluronic™ F-127 to the culture medium
4,5,13	Fluorescence intensity is low	Background fluorescence of the culture medium	Use Phenol red-free medium or buffer solutions during probe loading and observation
4,5	Basal fluorescence intensity is not constant	Laser power unstable	Wait 20~60 min before image acquisition
		Temperature unstable	Incubate cells at 37°C during the entire experimental procedure
10,13,27	FR measurements are not consistent	Cellular GSH concentration is affected by thermal stress	Maintain cells at a constant temperature (4°C) until flow cytometer analysis