JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry

Abstract

Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.

Figure 1

Standard methods for JC-1 detection are not optimal. L1210 cells were first incubated with 1 μM valinomycin or DMSO (control) for 30 min at room temperature (RT) and then stained with 2.5 μM JC-1 for additional 15 min (RT). Valinomycin and mock-treated cell populations were analyzed either separately or mixed by the Gallios Flow Cytometer (Beckman-Coulter), using a 488 laser, following standard protocols provided by JC-1 manufacturers. Data were processed by FlowJo v7.6.4. FL2 (585/42)−FL1 (525/50) subtraction (compensation) are shown in percentages.

Figure 2

Emission/excitation spectra of JC-1 monomers and aggregates reveals the potential use of 405 nm wavelength to specifically detect J-aggregates. JC-1 was dissolved in the presence of 0.1 (a), 35 (b) or 15% (c) DMSO to a final concentration of 2.5 μM. Emission spectra (a–c) at 405 (black) and 488 nm (gray), and excitation spectra (d) at 530 (black) and 595 nm (gray) were determined by Aminco Bowman Series 2 luminescence spectrometer. Excitation spectra of JC-1 at 0.1% DMSO (aggregates) and 35% DMSO (monomers) were determined separately (d). The black arrow indicates excitation at 405 nm. Also presented are the normalized emission (405 nm) and excitation (530nm) spectra for better visualization of the weaker signal (dotted line). The normalization factor for the emission spectra is the ratio between the fluorescent intensities measured at 595 nm of the 405 and the 488 nm-excited JC-1, and was determined to be 16.1 (a), 23.6 (b) and 13.3 (c). The normalization factor for the excitation spectrum of the 530 nm-emitted signal (=8.42) is the ratio between the 488 nm-excited fluorescence intensities at 530 nm and 595 nm (d).

Figure 3

Excitation at 405 nm specifically detects J-aggregates in the mitochondria. (a) HeLa cells were stained with 2.5 μM JC-1 (15 min, RT). Cells were excited at 405 or 488 nm and imaged by an upright Leica SPE confocal microscope (Leica, Wetzlar, Germany) ( × 40 water deep lens), using a single spectral detector. Emission spectra ranging from 513–641 nm in 10 nm intervals are depicted. (b) HeLa cells were grown in 35 mm glass-bottom dishes and stained with JC-1 (see a). Cells were imaged by Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany) using × 63 lens and the 405, 488 and 561 nm lasers. Filters used: BP 505-550 for the green channel and BP 575-630 for the red channel.

Figure 4

Excitation at 405 nm, like 561 nm, is optimal for flow cytometric analyses of J-aggregates. L1210 cells were incubated with DMSO (control) or 1 μM valinomycin for 30 min at RT and then stained with 2.5 μM JC-1 for additional 15 min. The control and valinomycin treated cell populations were mixed and analyzed by the LSRII flow cytometer (Becton Dickinson) using 405, 488 and 561 lasers, and 525/50 and 585/42 nm filters. Data were processed using FlowJo v7.6.4. Emission intensities at 585/42 were plotted also as histograms. The 405-nm-excited signal (grey) was normalized by a factor of 1.5-fold for better visualization.