*4.6. Measurement of Mitochondrial Membrane Potential (MMP)*

Changes in MMP were determined by the mitochondrial specific, incorporation of a cationic fluorescent dye Rhodamine-123 (Rh-123) [67]. In a typical experiment, the seeded cell in 96- well culture plates were treated with DEHP alone or combined to HE for 24 h. Then cells were rinsed with PBS and 100 μL of Rh-123 (1 μM) in PBS was added on the plates. Cells were incubated (37 ◦C, 5% CO2) for 15 min. Next, the PBS solution containing non-uptaken Rh-123 was washed and replaced by fresh PBS and estimated by fluorimetric detection. The results were expressed as the percentage of uptaken Rh-123 fluorescence relative to the fluorescence measured from negative control cells.

#### *4.7. Cell Death Induced by DEHP*

To distinguish apoptotic versus necrotic cells, Annexin V/propidium iodide (AnnV/PI) double staining was performed. PI in combination with FITC-AnnV permit to discriminate between viable (AnnV-/PI-), early apoptotic (AnnV+/PI-), and late apopto-tic/necrotic (AnnV+/PI+) cells. The Annexin V assay was performed following the manufacturer's instructions (Annexin V-FITC kit, Bender MedSystems, Vienna, Austria). Fluorescence of at least 5000 cells was analyzed by flow cytometry.

#### *4.8. Preparation of Mitoplasts*

PC12 cells were seeded on 6-well culture plates (Polylabo, France) at 1 <sup>×</sup> 105 cells/well for 24 h of incubation, after, the cells were incubated with DEHP (85 μM) alone or combined to HE (1 μM), for 24 h at 37 ◦C PC12 cells were collected by trypsinization, pelleted by centrifugation at 500× *g* and resuspended in PBS (pH 7.4). The cell suspension was exposed for 10 min on ice to 2 mg of digitonin/mg cellular proteins. The mitoplast fraction, obtained by digitonin cell disruption, was pelleted at 14,000× *g* and resuspended in PBS [45].
