Volume 56, Issue 5, Pages 1214-1216 (May 2012) 2′,7′-Dichlorofluorescein is not a probe for the detection of reactive oxygen and nitrogen species  Megan J. Reiniers  Journal of Hepatology  Volume 56, Issue 5, Pages 1214-1216 (May 2012) DOI: 10.1016/j.jhep.2011.10.012 Copyright © 2011 European Association for the Study of the Liver Terms and Conditions

Fig. 1 DCF is not a probe for the detection of reactive oxygen and nitrogen species. (A) Chemical structures of DCFH2-DA and derivates at physiological pH. DCFH2-DA (non-fluorescent at 523nm) is deacetylated (gray marquees, left panel) to DCFH2 by cytosolic esterases. DCFH2 (non-fluorescent at 523nm) undergoes a two-electron oxidation (gray marquees, right panel) in the presence of ROS/RNS to yield DCF. DCF has an absorption maximum at 503nm and an emission maximum at 523nm in aqueous solution. (B) Uptake of DCF by HepG2 cells. Cells were grown on fibronectin-coated coverglass bottom dishes in William’s E (WE) medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% l-glutamine, 0.1% insulin, and 0.2% hydrocortisone. Following 1h incubation in PBS or 50μM DCF in PBS at 37°C, 5% CO2, cells were washed and imaged by confocal microscopy at λex=496nm and λem=525±25nm. PC, phase contrast; FL, fluorescence; OV, overlay. (C) Quantification of DCF uptake by HepG2 cells as a function of DCF concentration. Cells were cultured on 24-well plates in supplemented WE medium as described above, washed, and incubated with 0–50μM DCF in unsupplemented WE medium for 1h at 37°C, 5% CO2. Following incubation, cells were washed and cellular DCF fluorescence was read with a microplate reader at λex=460±40nm and λem=520±20nm. Fluorescence emission intensity was normalized to protein content. Statistical analysis was performed using a one-way ANOVA and Dunnetts’s multiple comparison test following confirmation of normal distribution of data with a D’Agostino and Pearson omnibus test. (D) Superfusion of murine liver with DCF.The livers of adult male C57BL/6 mice (n=3) were superfused with 60μl of 200μM DCF in PBS for 45min. Subsequently, a punch biopsy of the superfused liver section was histologically processed and imaged by confocal microscopy. DCF fluorescence (λex=496nm and λem=525±25nm) is shown in green and TO-PRO 3 (λex=633nm and λem=670±30nm) was used as a clear stain (red fluorescence). (E) Oxidation-dependent DCF fluorescence kinetics. A cuvette containing 20μM DCF and 2mM Fe(II)SO4 in a 0.2M Na2HPO4/0.1M citric acid buffer (pH=6 or pH=7) (1485μl total volume) was placed in a temperature-controlled (20°C) spectrofluorometer set to λex=500±5nm and λem=523±5nm. During time-based acquisition, 15μl of H2O2 (35.6mM final concentration) or an equivalent volume of MilliQ was added at t=1min to the reaction mixture under continuous stirring. Addition of H2O2 resulted in the production of OH. Results represent the mean fluorescence emission values from n=3 experiments per group. [This figure appears in color on the web.] Journal of Hepatology 2012 56, 1214-1216DOI: (10.1016/j.jhep.2011.10.012) Copyright © 2011 European Association for the Study of the Liver Terms and Conditions