Cytochrome c antibody

MG-63 cells (3
× 10⁶/plate) were incubated with 2 μM DOX or DOX ⊂ 23a for either 24 or 48 h, and then, the mitochondrial
and cytosolic fractions were isolated by the differential centrifugation
method as previously described. The cytosolic release of cytochrome c was determined by Western blot of 5 μg of cytosolic
and mitochondrial fractions, using cytochrome c antibody
(Santa Cruz Biotechnology). Human GAPDH determination with GAPDH antibody
(Santa Cruz Biotechnology) was used as a positive control.

HepG2 and Huh7 cells were plated on 18-mm cover glasses for 24 h and then treated with LAC117 (100 μg/mL). A mitochondrion-specific dye (MitoTracker Red FM: Molecular Probes Inc., Eugene, OR, cat.n.M22426) was added and incubated for another 30 min. The media were removed, and the cells were washed with PBS and fixed with an acetone: methanol solution for 5 min at − 20 °C. The fixed cells were washed with PBS for several times and incubated with cytochrome c antibody (Santa Cruz Biotechnologies, cat.n.13156) overnight at 4 °C. Subsequently, after washing with PBS several times, the cells were incubated with a mouse fluorescenct-labeled secondary antibody (1:100, Vector Laboratories, Burlingame, CA, cat.n.TI-2000) for 1 h at room temperature. The cells were stained with DAPI to visualize the nuclei. Finally, the cells were covered with a fluorescent mounting solution (Dako, Carpinteria, CA, cat.n.REF3023) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Cells at a density of 3 X 10⁴ were grown in 0.2% gelatin coated coverslips in 35 mm plates. The 10 μM artemisinin treated cells were washed with ice-cold 1X PBS, fixed with methanol:acetone (1:1) and kept at -20 °C for 30 min-1 h. The cells were then blocked with blocking buffer [0.1% (w/v) bovine serum albumin, 0.3% (v/v) Triton™ X-100 in 1X PBS] for 2 h and then incubated with primary antibodies [Cytochrome c antibody (1:500, Santa Cruz), β catenin (1:5000)] overnight at 4 °C. Next day the cells were washed with TBST (1X TBST: 50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20.), then incubated with flourocrome conjugated anti-mouse antibody (1:1000, Alexa Fluor® 594, Life Technologies) for 1 h. The cells were then washed with TBST and further incubated with DiOC6 (3,3′-Dihexyloxacarbocyanine Iodide), a mitochondrial stain (1:1000, Life Technologies). Finally the coverslip was mounted on a slide using Prolong ® Gold Antifade Reagent (Life Technologies) and the images were captured using confocal microscope (Leica Microsystems CMS GmBH, Mannheim, Germany) using LAS AF application suite (Leica Application Suite Advanced Fluorescence).