Mitochondrial membrane potential assay kit

Cellular changes in the MMP were assessed using a JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbo cyanine iodide) Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Cells were exposed to drug(s) for 48 h and 0.5 mL of cell suspension was aliquoted into 5-mL tubes. Diluted (1:10 with cell growth medium), 4 μL MMP-sensitive fluorescent dye JC-1 reagent was added to each tube, incubated at 37°C for 20 min, and immediately analyzed by flow cytometry (λ_ex = 488 nm) using the 530-nm (FL-1 channel, green) and 585-nm (FL-2 channel, red) band-pass filters simultaneously. Healthy cells with functional mitochondria and high MMP exhibit red fluorescence (aggregated JC-1), whereas cells with disrupted mitochondria and low MMP show green fluorescence (monomeric JC-1). The ratio of monomeric/aggregated JC-1 was then calculated.

Mitochondrial membrane potential (ΔΨm) was assessed using the Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbour, MI, USA). The cytofluorimetric cationic dye JC-1 enters to the mitochondria and subsequently changes its fluorescent characteristics based on its aggregation activity. In healthy cells with a high ΔΨm, JC-1 forms complexes, emitting a red fluorescent light. However, in cells with a low ΔΨm the JC-1 dye remains as a monomer, followed by the emission of a green fluorescent light. The analysis was performed using the Glomax Multi+ combined spectro-fluoro-luminometer (Promega) on dark 96-wells plates. The resulting ΔΨm was expressed as the rate of JC-1 complexes to JC-1 monomers [68 (link)].

In order to have a closer look on the sperm mitochondrial behaviour following exposure to NTP, the mitochondrial membrane potential (ΔΨm) was examined with the Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). The protocol takes advantage of the fluorescent JC-1 cationic dye, which binds to the mitochondria and adjusts its fluorescent features depending on its aggregation. In the case of cells with healthy mitochondria with a high ΔΨm, JC-1 complexes emerge with red fluorescence. In the cells with disrupted mitochondria exhibiting a low ΔΨm, the JC-1 dye remains in its monomeric form with green fluorescence.
One million cells were transferred into Eppendorf tubes, the volume of the cell suspension was adjusted to 100 μL with PBS and subsequently stained with 5 μL JC-1 working solution. Following incubation (20 min, 37 °C, dark conditions) the stained suspensions were washed twice with PBS, transferred to a black opaque 96-well plate and the fluorescence intensity for the JC-1 monomers and polymers was quantified using a combined spectro-fluoro-luminometer (Glomax Multi⁺; Promega Corporation, Madison, WI, USA). The resulting ΔΨm is expressed as the ratio of JC-1 complexes to JC-1 monomers (red/green ratio) [67 (link)].

Mitochondrial membrane potential was measured using FACS and TMRE (tetramethylrhodamine, ethyl ester) mitochondrial membrane potential assay kit (Cayman Chemical, Ann Arbor, MI, USA). Cells were plated in 6-well plates and treated with and without glucose and 20 µM compound 6 or vehicle (DMSO). TMRE was added to the media to a final concentration of 500 nM and incubated for 20 min at 37 °C. Cells were washed with PBS and harvested using 200 µL of trypsin. Cells were collected with 800 µL of 0.2% BSA Containing PBS and fluorescence was measured using FACS.

Mitochondrial membrane potential was measured using TMRE (tetramethylrhodamine, ethyl ester) mitochondrial membrane potential assay kit (Cayman Chemical, Ann Arbor, MI, USA) and mitochondrial mass was measured using MitoTracker™ Green FM (Thermo Fisher Scientific). Cells were plated in 6-well plates and grown in complete medium or glucose starved for 24 h. TMRE was added to the media to a final concentration of 500 nM and incubated for 20 min at 37 °C. Cells were washed with PBS and harvested using 200 µL of trypsin. Cells were collected with 800 µL of 0.2% BSA containing PBS and fluorescence was measured using flow cytometry.