Jc 1 mitochondrial membrane potential detection kit

The mitochondrial membrane potential was measured using the JC-1 mitochondrial membrane potential detection kit (Cayman Chemical; Ann Arbor, Michigan, USA). The JC-1, a lipophilic cationic probe, accumulates in the mitochondrial matrix based on the membrane potential. A. lugdunensis (900 μL) were seeded in duplicate on a 24-well microtiter plate from a stock solution of 1 X 10⁵ cells/mL. Twentyfive μg/mL and 50 μg/mL T. nucifera solutions were prepared and added into each different well except the control group. After incubation for 24 h at 26°C, the cells were incubated for 24 h and centrifuged at 1500 rpm for 10 min, and then washed and resuspended in JC-1 buffer twice. The cell pellet was mixed with 100 μL buffer, and 10 μL of JC-1 was added. After incubation for 30 min at 26°C, the cells were centrifuged at 1500 rpm for 10 min. The cells were then analyzed using confocal fluorescence measurement.

The collapse of an electrochemical gradient across the mitochondrial membrane during PCD induction in treated amoebae was detected with the JC-1 mitochondrial membrane potential detection kit (Cayman Chemicals, Vitro SA, Madrid, Spain). Amoebae were incubated with the IC₉₀ of laurinterol for 24 h and the experiment was carried out by following the manufacturer’s recommendations. Images were captured using an EVOS FL Cell Imaging System AMF4300, Life Technologies, Madrid, Spain. The obtained staining pattern allowed the identification of two groups in a cellular population: live cells will show only red fluorescence; cells with low mitochondrial potential, (undergoing PCD) will show a higher level of green and red fluorescence.

We examined the loss of mitochondrial membrane potential (MMP) in response to KL-21 in 232B4 cells with the JC-1 Mitochondrial Membrane Potential Detection Kit (Cayman Chemicals, USA). Briefly, the cells (1x106 cells/2 mL), induced to undergo apoptosis, were collected by centrifugation at 180 x g for 10 min. Supernatants were removed and pellets were homogenized with 300 µL of medium, and then 30 µL of JC-1 dye was added to the cells and the cells were incubated at 37 °C in 5% CO2 for 30 min. They were centrifuged at 400 x g for 5 min, supernatants were removed, and 200 µL of assay buffer was added to the pellets and vortexed. This step was then repeated. Afterwards, all pellets were homogenized with 320 µL of assay buffer and 100 µL from each sample was added to a 96-well plate as triplicates. In healthy cells, the aggregate red form has absorption/emission maxima of 560/595 nm, whereas in apoptotic cells, the monomeric green form has absorption/emission maxima of 485/535 nm. The plate was read in these wavelengths with a fluorescence ELISA reader (Thermo Varioskan Spectrum, Finland). The ratio of fluorescent intensity of JC-1 monomers to fluorescent intensity of JC-1 aggregates was calculated for each concentration as well as the untreated control sample. Relative changes in cytoplasmic/mitochondrial JC-1 were then determined [16 (link)].