Heather Mary Brown, PhD | Application Scientist | Enzo Life Sciences
Matthew Hammer | Applications Scientist | Molecular Devices
Cheryl L. Bell, PhD | Field Applications Scientist | Molecular Devices
Introduction
From studying disease to evaluating drug and environmental compound-induced toxicity, cell-based assays aimed at monitoring cellular health often focus on mitochondria. Mitochondrial function is a critical indicator of overall cell health, highlighted by the association of mitochondrial dysfunction and a variety of diseases including Parkinson’s Disease, Alzheimer’s Disease, heart failure, ischemic diseases, and cancer.1 Additionally, the pathogenesis of rare diseases can be driven by mutations in genes coding for mitochondrial proteins or in mitochondrial DNA.1
Evaluating mitochondrial function is critical throughout drug development and testing. This is exemplified by the development of therapeutics to restore mitochondrial function, and the development of chemotherapeutics designed to target mitochondrial dysfunction in cancer.3 Furthermore, assessment of mitochondrial function is a vital step in preclinical drug safety assessment since mitochondrial dysfunction can be triggered by drugs and in turn, result in adverse effects on cellular and tissue health.
One sensitive and informative method to assess mitochondrial function is measuring mitochondrial membrane potential (MMP) to monitor changes in the metabolic activity of mitochondria. Mitochondrial membrane potential is a crucial tool for the detection of mitochondrial depolarization during apoptosis, the testing of multi-drug resistant cells, and the assessment of mitochondrial function for a variety of toxicity screens.1-2 Evaluation of MMP is commonly performed with the use of lipophilic, cationic fluorescent dyes. The MITO-ID® membrane potential dye exhibits changes in its fluorescence depending on the status of mitochondrial membrane potential. Specifically, it exists as a greenfluorescent (FITC, 530 nM) monomer in the cytosol regardless of MMP, and aggregates inside healthy, energized mitochondria to emit orange-fluorescence (TRITC, 570 nm). Upon disruption of MMP, the MITO-ID dye exits the depolarized mitochondria, resulting in low to no orange fluorescent aggregates and an accumulation of the dye monomers in the cytosol.
We performed two mitochondrial toxicity assays utilizing the MITO-ID dye to assess changes in mitochondrial membrane potential in response to compound treatment. Assays were performed on a suspension cancer cell line, SJK, and an adherent cancer cell line, U2OS, to demonstrate the utility of MITO-ID dye to assess MMP in multiple cell types and the robust imaging and analysis capabilities of the ImageXpress Pico® Automated Cell Imaging System.
Materials
- SJK cells (ATCC, cat. #CRL-1644™)
- U2OS cells (ATCC, cat. #HTB-96™)
- MITO-ID Mitochondrial Membrane potential detection kit (Enzo Life Sciences, cat. #ENZ-51018)
- Hoechst 33342 (Enzo Life Sciences, cat. #ENZ-52401)
- 96 well, clear suspension culture microplate (Greiner Bio-One, cat. #655185)
- 96 well black wall, μClear® microplate (Greiner Bio-One, cat. #655090)
- ImageXpress Pico Automated Cell Imaging System with the CellReporterXpress Image Acquisition and Analysis Software
Methods
Assaying mitochondrial toxicity in suspension and adherent cancer cells
Suspension Cells
SJK cells were plated into a 96-well suspension cell plate at 10,000 cells per well. The cells were treated for 30 minutes with a 1:3 dilution of CCCP, from 2.5 nM to 50 μM. Compound treatments were performed in triplicate. After treatment, the cells were spun down, supernatant was removed, and the cells were resuspended and washed in 1X Assay Buffer. The cells were centrifuged once more, and the supernatant was removed prior to the addition of the MITO-ID and Hoechst 33342 staining solution. Hoechst was prepared at 1 μL/mL in 1X assay buffer, and the MITO-ID Membrane potential detection reagent was prepared at 10 μL/mL.
The cells were incubated with the staining solution for 15 minutes at room temperature in the dark, and then imaged immediately on the ImageXpress Pico system at 40X magnification. Forty-two sites per well were acquired in transmitted light in combination with the DAPI, FITC, and TRITC fluorescent channels. Images were collected at different depths within the well to ensure that all the cells in suspension were in focus. This was accomplished by capturing a stack of images, called a Z-stack, and the stack of images were combined into a single 2D projection image on-the-fly. The Z-stack acquisition was performed with a small stack of five planes and a focus step size of 2 μm. The best focus 2D projection images from the Z-stacks were generated and utilized for analysis.
Adherent Cells
U20S cells, were seeded at 8,000 cells per well in a black wall, clear bottom 96-well microplate. The plates Figure 2. Evaluation of mitochondrial membrane potential changes in SJK cells in response to treatment with CCCP. SJK cells were stained with the MITO-ID Membrane potential dye, which A, C) fluoresces green in the cytosol regardless of MMP and forms orange aggregates in polarized mitochondria with intact MMP. B,D) The cells were analyzed with the 3-channel Multi-Wavelength Cell Scoring Application Module in CellReporterXpress software. Cells were segmented based on their Hoechst 33342 nuclear staining and scored positive for FITC (red segmentation mask), positive for TRITC (blue segmentation mask), positive for both FITC and TRITC (pink segmentation mask), and negative for both FITC and TRITC (grey segmentation mask). Cells that scored positive for TRITC only and FITC plus TRITC were counted as cells with active mitochondria. Multiparametric readouts were generated from the analysis, including E) percent of cells positive for TRITC signal, F) average TRITC cytoplasmic intensities, and G) average FITC cytoplasmic intensities. Replicates were averaged, and the curves produced EC50 values of 0.639 μM, 0.265 μM, and 0.05 μM respectively. were incubated overnight at 37°C, 5% CO2. The cells were treated with either CCCP or Antimycin A in a 1:3 dilution starting at 50 μM CCCP and 100 μM Antimycin A. Compound treatments were performed in quadruplicate. After one hour of treatment, the compound solution was removed from the wells and the cells were washed once in 1X Assay Buffer. The MITO-ID staining solution was prepared at 10 μL/mL in 1X assay buffer following the manufacturer’s protocol, and Hoechst 33342 was added at 1 μL/mL. The plate was incubated at room temperature for 30 minutes with the staining solution, then washed with HBSS (2X). Images were acquired with the ImageXpress Pico system using the 20X objective, capturing six sites per well with an on-the-fly stitching protocol. The DAPI, FITC, and TRITC fluorescent channels were utilized in the acquisition with the following exposure times respectively, 10 ms, 200 ms, and 200 ms.
Results
Quantifying the phenotypic effects of CCCP on SJK cell mitochondrial membrane potential
SJK cells, mouse myeloma B lymphocyte cell line, were treated with a potent inhibitor of oxidative phosphorylation, CCCP (a proton ionophore), and then stained post- treatment with the MITO-ID Membrane potential detection kit and Hoechst 33342 nuclear dye. Differences in the fluorescence emission of the MITO-ID dye were visualized and quantified with automated imaging and analysis. In energized cells the dye existed as a green (FITC) fluorescent monomer in the cytosol, while forming aggregates that fluoresces orange (TRITC) in active mitochondria with intact MMP (Figures 1 and 2). Analysis of mitochondrial membrane potential was performed with the 3-channel Multi-Wavelength Cell Scoring Application Module in the CellReporterXpress® Image Acquisition and Analysis Software. Cells were counted based on the nuclear stain and the cells were scored positive or negative for FITC signal, TRITC signal, or both fluorescent signals. SJK cells that expressed both FITC and TRITC fluorescent molecules as well as only TRITC aggregates were counted as positive for intact mitochondrial membrane potential (Figure 2A-E). Multiparametric readouts including number and percent of cells positive for one, both, or no fluorescent markers, cellular and nuclear area measurements, and intensity measurements for the two fluorescent markers (FITC and TRITC) were generated from the analysis.
Figure 1. Representative 40X images of untreated SJK cells captured on the ImageXpress Pico. Images were captured at different planes with a Z-stack, and the Z-stack images were collapsed into a single 2D projection image in the CellReporterXpress software. The best plane 2D projection was generated for the A) transmitted light channel, while the best focus 2D projection was utilized for the fluorescent channels: B) DAPI channel, Hoechst 33342 stained nuclei, C) FITC channel, MitoID fluorescing green in the cytosol, and the D) TRITC channel, MitoID fluorescing orange in active mitochondria.
There was a striking dose-dependent effect on MMP marked by the decreasing percent of cells positive for orange TRITC aggregates in polarized mitochondria as the concentration of CCCP increased (Figure 2E). The decrease in overall TRITC signal intensity in higher concentrations of CCCP combined with an increase in cytosolic green intensity, also indicated loss of mitochondrial function (Figure 2F). At the highest three concentrations, the green FITC signal began to decrease in the cells demonstrating the toxic effects of these concentrations of CCCP.
Figure 2. Evaluation of mitochondrial membrane potential changes in SJK cells in response to treatment with CCCP. SJK cells were stained with the MITO-ID Membrane potential dye, which A, C) fluoresces green in the cytosol regardless of MMP and forms orange aggregates in polarized mitochondria with intact MMP. B,D) The cells were analyzed with the 3-channel Multi-Wavelength Cell Scoring Application Module in CellReporterXpress software. Cells were segmented based on their Hoechst 33342 nuclear staining and scored positive for FITC (red segmentation mask), positive for TRITC (blue segmentation mask), positive for both FITC and TRITC (pink segmentation mask), and negative for both FITC and TRITC (grey segmentation mask). Cells that scored positive for TRITC only and FITC plus TRITC were counted as cells with active mitochondria. Multiparametric readouts were generated from the analysis, including E) percent of cells positive for TRITC signal, F) average TRITC cytoplasmic intensities, and G) average FITC cytoplasmic intensities. Replicates were averaged, and the curves produced EC50 values of 0.639 μM, 0.265 μM, and 0.05 μM respectively.
Multiparametric analysis of mitochondrial membrane potential in U2OS cells
Adherent U2OS cells treated with known inhibitors of oxidative phosphorylation, CCCP and Antimycin A (mitochondrial respiratory chain complex III inhibitor) demonstrated a striking dose-dependent effect on mitochondrial membrane potential. This was evaluated via the Mitochondrial Analysis Module in CellReporterXpress software, which was utilized to quantify total cell number based on nuclear staining as well as the number of orange TRITC fluorescent aggregates indicative of active mitochondria with intact MMP (Figure 3). Loss of mitochondrial membrane potential was demonstrated by a decrease in the average TRITC aggregate count per cell in addition to the decrease in the TRITC signal intensity of the aggregates as the concentration of both compounds increased (Figure 4).
Figure 3. Effect of oxidative phosphorylation inhibitors on U2OS cell mitochondrial membrane potential. Cells were treated with dilutions of CCCP or Antimycin A and then stained with the MITO-ID membrane potential dye and Hoechst 33342. (Top panel) The green fluorescent cytosolic dye was imaged with the FITC channel, the orange aggregates with the TRITC channel, and blue nuclei with the DAPI channel. (Bottom Panel) Analysis segmentation masks were generated by the Mitochondria Analysis Module in CellReporterXpress software where the nuclei mask was displayed in green and the TRITC fluorescent aggregate mask was shown in white.
Figure 4. Analysis of orange, TRITC fluorescent aggregates accumulation in intact mitochondria of U2OS cells in response to compound treatment. Multiple readouts were generated by the Mitochondrial Analysis Module in CellReporterXpress software, which quantified cells based on their Hoechst 33342 nuclear stain and counted the number of TRITC aggregates per cell. (A) Average granule count and (B) average TRITC intensity for the aggregates were displayed here. 4-parameter logistic curve fits were applied and generated EC50 values of 0.297 μM (Antimycin A) and 0.303 μM (CCCP) for the average TRITC aggregate count, and EC50 values of 0.656 μM (Antimycin A) and 0.574 μM (CCCP).
Conclusion
We demonstrated the effective use of a highly sensitive cationic fluorescent dye (MITO-ID) to visualize and analyze mitochondrial membrane potential changes by automated imaging. Changes in mitochondrial membrane potential were visualized through the change in emission spectra from the dye, as well as the generation of multiparametric readouts. In summation, the ImageXpress Pico system in combination with the MITO-ID Membrane potential detection kit is evidenced to be a paramount tool for monitoring changes in mitochondrial membrane potential in both suspension and adherent cancer cell lines. These data also highlight the utility of this mitochondrial membrane potential assay for monitoring mitochondrial function in preclinical drug safety assessment as well as chemical/environmental toxicity screening.
References
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