fluorescence intensity quantification of transfected hela cells using imagej

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

The mitochondrial network is a series of interconnected organelles that play a crucial role in energy production1, innate immunity2,3, and cell signalling4,5. Mitochondrial dysregulation has been associated with neurodegenerative diseases such as Parkinson&#39;s disease (PD)6,7. PD is a progressive neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra that impacts nearly 10 million people worldwide8. PD has been genetically linked to mitophagy, a mitochondrial quality control pathway necessary for maintaining cellular homeostasis that selectively removes damaged mitochondria9,10. Studies have identified multiple independent mitophagy pathways, including FUN14 domain containing 1 (FUNDC1)-mediated mitophagy, Bcl-2 interacting protein 3 (BNIP3)-facilitated mitophagy, NIX-dependent mitophagy, and the well-characterized PTEN-induced kinase 1 (PINK1)/Parkin-regulated mitophagy10,11. PINK1 (a putative kinase) and Parkin (an E3 ubiquitin ligase) work in tandem to phospho-ubiquitinate damaged mitochondria, which drives the formation of autophagosomes that engulf the damaged organelle and fuse with lysosomes to initiate degradation12,13,14,15,16. Mutations in Parkin have been associated with PD-linked phenotypes such as neurodegeneration via the loss of dopaminergic neurons17,18.
Here, a protocol is described in which HeLa cells, routinely used immortalized cells derived from cervical cancer, are used to investigate the role of Parkin in maintaining mitochondrial network health. HeLa cells express negligible levels of endogenous Parkin and therefore require exogenous Parkin expression19. To study the role of Parkin in mitochondrial network health, HeLa cells are transfected with either wild-type Parkin (ParkinWT), a Parkin mutant (ParkinT240R), or an empty control vector. ParkinT240R is an autosomal recessive juvenile parkinsonism mutation that affects Parkin E3 ligase activity, significantly reducing the efficiency of the mitophagy pathway20. HeLa cells are subject to mild (5 &#181;M) or severe (20 &#181;M) concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. Treatment with severe concentrations of CCCP is routinely used to induce Parkin-mediated mitophagy in various cell lines, such as HeLa and COS-7 cells21,22,23.
Following treatment, the protocol employs live imaging of the mitochondrial network using two currently available mitochondrial-targeted fluorescent dyes. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) is a cationic dye that fluoresces based on mitochondrial membrane potential24, while MitoSOX is a mitochondrial superoxide indicator where the fluorescence intensity is a function of superoxide concentration25. Finally, the outlined protocol uses a fluorescence-based quantification and simple workflow to effectively quantify the mitochondrial membrane potential and superoxide levels with minimal scope for user bias. Although this protocol was designed to study mitochondrial function in HeLa cells, it can be adapted for additional cell lines and primary cell types to quantitively characterize mitochondrial network health.

Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells  

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

This research focuses on mitochondria dysregulation in neurodegenerative diseases. We believe that this research can be used to understand potential causes for disease onset that could lead to therapeutics combating neurodegenerative diseases like Parkinson's disease and ALS. Techniques such as super resolution microscopy like STED and SIM or expansion microscopy have improved the ability to accurately understand protein distribution within individual organelles and mitochondria distribution throughout the cell.Results of this technique can be used as a starting point to study the effect of Parkinson's disease linked mutations on mitochondrial turnover and begin to understand the importance of regulating reactive oxygen species levels and mitochondrial membrane potential to maintain neuronal health. Our laboratory aims to mechanistically characterize individual mitochondrial quality control pathways to understand the interplay between these pathways. By having insights into pathway dynamics, one can understand how mitochondria are maintained and how mitochondrial dysregulation contributes to neurodegenerative disease onset.

Watch this Scientific Journal Video about Fluorescence Intensity Quantification of Transfected HeLa Cells Using ImageJ at JoVE.com

Fluorescence Intensity Quantification of Transfected HeLa Cells Using ImageJ  

To measure the fluorescence intensity of the transfected HeLa cells in ImageJ software, click file and select open. In the dialogue box, select the imaging files for the experiment and click okay. Once the bio-formats import options window appears, select split channels and click okay.In tetramethylrhodamine, ethyl ester, perchlorate, or TMRE experiments, YFP is the first channel, MitoTracker is the second channel and TMRE is the third channel. Adjust the brightness by selecting image, then adjust and then the brightness. Next, load the saved region of interest or ROI by clicking analyze, then tools, then ROI manager.In the ROI manager, click more, then open from the list and select the saved ROI. Then measure the fluorescent intensity of five random regions in a single cell by selecting the saved ROI from the ROI manager and moving the ROI to a random location within a cell. To measure the fluorescence intensity, press M on the keyboard and repeat this with four additional non-overlapping regions.When a dialogue box with the area and mean gray values appears, copy and paste the values into a spreadsheet for analysis. The results for the TMRE and MitoSOX fluorescence intensities showed that their treatment with the known uncoupling agent CCCP decreased the TMRE fluorescence intensity compared to the control conditions. In addition, 20 micromolar CCCP treatment induced superoxide production and increased the MitoSOX fluorescence intensity.In mild or five micromolar CCCP stress conditions, the expression of Parkin wild-type and Parkin T240R resulted in higher TMRE intensity compared to the empty YFP control vector. The MitoSOX intensity was lower in cells expressing Parkin wild-type and Parkin T240R compared to cells expressing the YFP control vector suggesting that Parking expression helps maintain the mitochondrial network health by preserving higher mitochondrial membrane potentials and low superoxide levels.

fluorescence-intensity-quantification-transfected-hela-cells-using

Research

JoVE Journal

Biology

2.8K Views.  Duke University Medical Center. To measure the fluorescence intensity of the transfected HeLa cells in ImageJ software, click file and select open. In the dialogue box, select the imaging files for the experiment and click okay. Once the bio-formats import options window appears, select split channels and click okay.In tetramethylrhodamine, ethyl ester, perchlorate, or TMRE experiments, YFP is the first channel, MitoTracker is the second channel and TMRE is the third channel. Adjust the brightness by selecting image,...