Cytochrome c Profiling ELISA Kit (ab110172)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.	-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.	-Unused well strips should be returned to the plate packet and stored at 4°C.

Upstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.
Protocol tips
-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.
Downstream tips
-Unused well strips should be returned to the plate packet and stored at 4°C.

Publication protocol

The Mitochondrial Fractionation kit (Active Motif, Inc., Carlsbad, CA, USA, Cat. No. 40015) was used to isolate mitochondrial and cytosolic fractions from cells under the manufacturer’s instructions. Briefly, cells were seeded at a density of 2.4 × 105 cells/well in 6-well plates and incubated for 24 h and then treated with or without diosmin or taurine for 48 h. The treated or untreated ARPE-19 cells were scraped and spun twice at 600× g for 5 min. Ice-cold 1× cytosolic buffer was added and the cell pellet was resuspended and incubated on ice for 15 min. Cells were homogenized and the lysate was spun twice at 800× g for 20 min. The resultant supernatant contained the cytosol, including mitochondria; the supernatant was spun at 10,000× g for 20 min to pellet the mitochondria. The mitochondrial pellet was washed and spun with 1× cytosolic buffer at 10,000× g for 10 min, and then lysed by adding Complete Mitochondria Buffer followed by incubation on ice for 15 min, the result of which was the mitochondrial fraction. At the same time, the supernatant was centrifuged at 16,000× g for 25 min. The centrifuged supernatant was the cytosolic fraction. The protein concentration was measured by using a Bio-Rad protein assay. After isolation of mitochondria and cytosolic fraction, the Cytochrome C ELISA kit (Abcam plc., Cambridge, MA, USA, Cat. No. ab110172) was used to measure the level of cytochrome c according to the manufacturer’s instructions.

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Manufacturer protocol

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