Jc 1 mitochondrial membrane potential assay kit

MOVAS and RAW 264.7 cells were cultured to confluence in a 96-well black culture plate as previously described in 100 μL of media per well. Cells were then treated with 1 μM Ang II, 1 μM Ang II plus 100 ng/mL RANKL-neutralizing antibody, 100 ng/mL RANKL-neutralizing antibody alone, or left untreated for 48 hours. Five wells per treatment condition were prepared. After 2 days of incubation, apoptosis was analyzed with the JC-1 mitochondrial membrane potential assay kit according to the manufacturer’s instructions (Cayman Chemical). Briefly, 10 μL of JC-1 staining solution was added to each well and allowed to incubate for 30 minutes at 37°C in a carbon dioxide incubator. The plate was then centrifuged for 5 minutes at 400 g, and the supernatant was removed. The wells were then washed twice with 200 μL of phosphate-buffered saline (PBS) and centrifuged for 5 minutes at 400 g, and the supernatant was removed. Last, 100 μL of PBS was added to each well, and the plate was read on a FlexStation 3 microplate reader. The fluorescence intensities of each well were recorded for the 535/595 nm and 485/535 nm excitation/emission pairs. Data are reported as the ratio of green fluorescence at 535 nm to red fluorescence at 595 nm.

JC-1 mitochondrial membrane potential assay kit (Cayman chemical, Ann Arbor, MI) was used for this assay, following the manufacture’s instruction. The cytofluorimetric, lipophilic cationic dye, 5,5′,6,6′-tetrachloro-1,1′3,3-tetracthylbenzimidazolylcarbocyanine idodide (JC-1), can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. AM cells were treated as above and an equal amount of dye was added. After 30-min incubation, fluorescence was measured using a fluorometer, using 560 nm excitation and a 595 nm emission filter for detecting healthy cells, and 485 nm excitation and a 535 nm emission filter for detecting dead cells (35 (link)).

Immediately after the short-term pyocyanin treatment or twenty-four hours after the last long-term pyocyanin treatment, MUTZ-3 cells were collected and stained with JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s protocol. Cells from the control groups were set aside for positive and negative CellROX^® controls as described above. JC-1 cells were sorted base on live cells, and processed according to the protocol used for MSCs, with the one substitution of CD34-PE to phenotype HPCs. The following day, at the completion of the RedOx staining protocol, cells were analyzed based on CD34 expression.

Cell health in the presence of the three metal ions was assessed using a JC-1 mitochondrial membrane potential assay kit (Cayman Chemical Company, Ann Arbor, MI). NSCs were cultured in the presence (10 nM - 100 pM) or absence (0 μM; controls) of metal solutions for 14 days as described above (n = 6 wells/ concentration/ metal), and the cultures processed with JC-1 staining solution as per vendor’s protocols. Cells were imaged using the Zeiss Axiovert A1 florescence inverted microscope, with healthy cells detected using a Texas Red filter and apoptotic cells detected with a FITC filter. Details are available in supplementary methods.