Peroxisomal oxidation of oleate was estimated following inhibition of oxidative phosphorylation using rotenone, Antimycin A and potassium cyanide in the reaction buffer and repeating the assay.
Twenty microliters of cells was mixed with 180 μl of the assay buffer (25 m M pNPP [Cat. No. N4645; Sigma], 1 m M zinc acetate in a 100 m M Tris buffer pH8) and the assay was performed in 96-well microtiter plates, measuring the rate of pNPP hydrolysis at 410 nm every 30 s for 2 h at 37°C.
The supernatant was discarded, and cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer and the TEM assay was performed following a previous study.
On the fifth day (48 hr after chemical treatment), the cells were lysed in 100 μL/well passive lysis buffer, and the luciferase assay was performed in accordance with the protocol of the Dual-Luciferase Reporter Assay System (Promega) using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany).
To estimate total ascorbate, 100 µL of dithiothreitol (DTT) and 200 µL of reaction buffer (150 mM potassium phosphate buffer) were added and the assay mixture was thoroughly mixed and incubated for 15 min at 25 °C; 100 µL of 0.5%% (w/v) N-ethylmaleimide was then added.
Finally, 10 µL mushroom tyrosinase (500 U/mL in phosphate buffer) was added and the assay mixture was then incubated at 27 °C for 10 min.
The cells were then incubated for 15 min in dark, 400 μL binding buffer was added and the assay was begun immediately using a FACScan flow cytometer (Becton Dickinson).
At 72 h after transfection, cells were lysed in 100-μl passive lysis buffer, and the dual luciferase assay performed according to the manufacturers protocol (Promega).
Equal volumes of cytosolic fraction and reaction buffer from the assay kit were mixed along with cathepsin B substrate Ac-RR-AFC and incubated for 1 h at 37°C.
Afterwards, the supernatant was recovered, further diluted (1/10) with the same buffer and the activity was assayed following the same procedure described above by mixing 50 µL with 575 µL of 1.1 X hexosaminidase substrate buffer.
