This protocol greatly maximizes the power of microscopy as a method to track mitochondrial dynamics within individual cells over long acquisition periods. Unlike time-lapse on a fixed group of cells, or on one cell at a time, normalization to a baseline for each measured criterion in each cell controls for the heterogeneity between cells. With a microscope equipped with automatized platform and viral vector adapted, we can use this protocol for every type of cells.
Begin by plating 20,000 to 25, 00 cells per square centimeters in multi-well dishes and store them at 37 degrees celsius under an atmosphere containing 5%carbon dioxide for the rest of the experiment. At eight days in vitro, add 0.6 pico grams of P24 antigen per cell of lentiviral vector coding for mitochondrial biosensor MitoTimer diluted in phosphate buffered saline. At 11 days in vitro, perform two washes with pre-warmed sterile PBS and add fresh astrocyte medium without phenol red.
Assess the Astrocytic mitochondrial system at least three to five days after the lentiviral infections with LV-G1 MitoTimer. Select five astrocytes per well with a mitochondrial network expressing sufficient levels of LV-G1 MitoTimer using a magnification of 40 times. Taking care to select astrocytes flat and large as possible and not located in clusters of cells.
Then capture fluorescence images using sequential excitation at 490 nanometers for the green channel and 550 nanometers for the red channel with green and red fluorescent signals and using a magnification of 150 times for each coordinate. Select the first frame for red and green channels for each image sequence by clicking on ND Processing and select frame. Then merge the red and the green channels by selecting conversions and merge channel.
To correct image shading, select pre-processing, and then auto shading correction. Apply the rolling ball algorithm by selecting pre-processing and rolling ball. To generate binary masks for each mitochondrion, select segmentation and threshold.
Remove any objects truncated by the border by selecting binary processing and touching border. Select measurement, and then object area, EEQ diameter, length, width, roughness, circularity, or elongation to measure surface area, diameter, length, width, roughness, circularity, or elongation respectively. Compose a group with these measurements and rename it as MOFO data.
Then select measurement, and then select mean intensity to measure the mean green and red intensities. And ratio to measure the red by green ratio. Now composer group with these measurements and rename it as ratio data.
Finally export the table to a CSV file by selecting reference and table to CSV, and then save the GA 3 script of analysis by selecting Save As.Begin by opening the tracking module in NIS and selecting view, analysis and tracking. Click on define new ROI. With the automatic detection tool, select 25 to 50 mitochondria on the first image of the image sequence, and then click on track auto detected ROIs analyze.
If necessary, delete the incorrect ROI tracks and export the table to a CSV file. Manually log transform the measurements before processing. For each analysis and timeframe manually normalize the resulting data by the mean obtained in the reference acquisition.
Then perform statistical analysis using a two way matched a Nova. Primary culture of astrocytes infected with LV-G1 MitoTimer exhibited reduced balanced and oxidized mitochondrial networks with different levels of fragmentation. Before hydrogen peroxide treatment, astrocytes expressing LV-G1 MitoTimer showed heterogeneous mitochondrial size in various green and red fluorescence intensities.
The mitochondrial morphology of astrocyte cultures was fragmented after six hours of incubation with hydrogen peroxide. It was even more apparent 12 hours after the treatment without their diameters with since sphericity reduction. Concerning redox state and turnover, three hours after a hydrogen peroxide treatment, the proportion of green mitochondria increased in astrocytes.
Concerning the dynamics and mobility three hours after treatment. All the criteria were transiently increased. This technique is suitable for many different questions.
For example, in the context of neurodegenerative disease, we're investigating the estrocytic toxicity of different molecules secreted in the brain.