Table 1.

Guidelines for proper use of fluorescent probes.

Fluorogenic probe	Advantages	Disadvantages	Recommendations
HE	Reacts with $O_{2}^{• -}$ to form a diagnostic marker product, 2-OH-E⁺. Reacts with other oxidants (e.g., ^•OH, ONOO⁻) to form E⁺ and dimers. Intermediate HE-derived radical does not react with molecular oxygen to form $O_{2}^{• -}$ and H₂O₂.	2-OH-E⁺ and E⁺ have similar fluorescence spectral characteristics. Further extraction and HPLC analyses are needed for proper identification.	Suitable for detection of intracellular $O_{2}^{• -}$ . Suitable for detection of intracellular oxidants (e.g., iron and H₂O₂, cytochrome c and H₂O₂). However, red fluorescence measurements using fluorescence or confocal microscopy will not be able to distinguish between E⁺ and 2-OH-E⁺. HPLC or other analytical measurements of products are crucial.
Mito-SOX or Mito-HE	Localizes into mitochondria. Reacts with $O_{2}^{• -}$ to form a diagnostic marker product, 2-OH-Mito-E⁺. Reacts with other oxidants (^•OH, ONOO⁻) to form Mito-E⁺ and dimers. Intermediate Mito-HE-derived radical does not react with O₂ to form $O_{2}^{• -}$ and H₂O₂.	2-OH-Mito-E⁺ and Mito-E⁺ have similar fluorescence spectral parameters. HPLC analyses of products are necessary for proper identification probe. Mitochondrial toxicity is a concern.	Suitable for detection of mitochondrial $O_{2}^{• -}$ . Suitable for detection of intracellular oxidants (e.g., iron and H₂O₂, cytochrome c and H₂O₂). However, the “red fluorescence” measurements alone will not allow distinction between the hydroxylated and the oxidized Mito-HE. HPLC or other analytical measurements of products are crucial.
DCFH-DA	Cell-permeable Easy to use. Responds to changes in intracellular iron signaling or enhanced peroxidase activity (see Ref. [36]).	Artifactual amplification of the fluorescence intensity via a redox-cycling mechanism involving an intermediate radical, DCF^•−.	Not suitable for measuring intracellular H₂O₂ or other oxidants. May be used as a redox indicator probe for uncovering new redox signaling mechanisms keeping in mind the various caveats.
DHR	Cell-permeable Easy to use. Responds to cogenerated ^•NO and $O_{2}^{• -}$ via a predictable radical chemistry.	Artifactual amplification of the fluorescence intensity via a redox-cycling mechanism involving an intermediate radical, DHR^•.	Not suitable for measuring intracellular ONOO⁻ or other oxidants. May be used as a nonspecific indicator for intracellular ONOO⁻ or oxidants derived from it.
Coumarin boronate	Reacts very rapidly and nearly stoichiometrically with ONOO⁻ to form a fluorescent product.	Further metabolism of the product (7-hydroxycoumarin) and possible excretion out of cells may be a hindrance.	Suitable for measuring extracellular ONOO⁻ formation. As this probe also reacts with H₂O₂ (albeit slowly), proper controls using appropriate inhibitors should be performed.
Amplex red	HRP/H₂O₂-dependent oxidation of Amplex red to resorufin is very efficient. The intermediate Amplex red-derived radical does not react with O₂ for form additional $O_{2}^{• -}$ and H₂O₂. The intermediate Amplex red-derived radical will react with $O_{2}^{• -}$ to decrease resorufin formation (see Ref. [37]). Reducing agents (e.g., ascorbate) and peroxidase substrates (nitrite anion) will inhibit resorufin formation.	Room light-mediated photochemical oxidation of resorufin in the presence of GSH, NAD(P)H, or ascorbate will greatly increase artifactual generation of H₂O₂. Amplex red-derived radical can rapidly react with $O_{2}^{• -}$ and inhibit product formation.	Suitable for measuring extracellular H₂O₂ under conditions limiting the secondary radical reactions induced by resorufin.