The overall goal of this procedure is to monitor changes in mitochondrial calcium concentration in live cells. This is accomplished by first transecting cells with the calcium indicator protein ratio metric peram, which is targeted to the mitochondrial matrix, one or two days following transfection. The fluorescence microscope and software is set up for live cell imaging.
Images of ratio metric peram expressing cells are then acquired upon addition of a calcium mobilizing agonist. The final step of the procedure is quantitative analysis of the acquired images. Ultimately, results can be obtained that show dynamic changes in mitochondrial calcium concentration through fluorescence microscopy of live cells expressing a ratio metric calcium indicator protein.
The main advantage of this technique over existing methods such as measuring calcium levels using synthetic calcium indicators is that ratio metric peram is genetically encoded and thus can be targeted to mitochondria or any other organal of interest. Hello, I'm Dr.Ascar, a postdoc in Dr.Benning's laboratory, and I will be demonstrating the procedure today To begin the protocol plate helo cells the night before, transfection on sterile glass cover slips placed in a standard six well culture plate at a density that will be approximately 70%con fluent for transfection the following day. The next day prepare DNA Lipectomy 2000 complexes per the manufacturer's instructions with four micrograms of metric peram mitochondrial expression vector, and 10 microliters of lipectomy 2000 diluted in 0.5 milliliters of optimum for each well to be transfected.
Next, replace the D-M-E-M-F-B-S HELOC culture media in each well with 1.5 milliliters of the same media without antibiotics. Add the LIPECTOMY solution to the DNA solution and mix gently after the complexes have formed. Add the DNA Lipectomy solution dropwise to each well to be transfected mixed by gently rocking the plate and incubate for four hours in a cell culture incubator at 37 degrees Celsius, 5%carbon dioxide following the incubation.
Replace the media with D-M-E-M-F-B-S containing antibiotics. Allow the cells to express metric peram for one to two days before imaging using forceps. Gently place a cover slip with HELOC cells into an appropriate imaging chamber with imaging solution.
Mount the imaging chamber in an inverted microscope stage. Next, use a 40 x or higher oil immersion objective and a wavelength of 380 nanometers. To find an area of interest containing one or more cells expressing ratio metric peram 380 nanometers is used here to identify the cells as ratio metric.
Peram is less resistant to photobleaching at this wavelength. Once an area of interest has been identified, set up the software for dual excitation at 495 and 380 nanometers. Next, acquire images sequentially by alternatively exciting at 495 and 380 nanometers.
Acquire images of baseline calcium levels for at least 30 seconds before application of agonist. Then apply the calcium mobilizing agonist via a profusion apparatus, noting the addition time for each agonist exposure times and acquisition intervals should be optimized to prevent photo bleaching while still allowing sufficient temporal resolution. Once the experiment is complete, images can be analyzed offline.
To begin analysis, select a region of interest within an empty area of the field to subtract background fluorescence if necessary. Next, export the 4 95 3 80 ratio measurements from the region of interest to Excel or similar graphing software. This panel of images shows the subcellular localization of ratio metric peram and mitochondria.
On the left is a live cell image of helo cells expressing ratio metric peram. In the middle is the fluorescent staining of the mitochondria and selective dye MIT tracker red CMX Ross. And finally on the right, the merged image shown here is a series of pseudocolor 4 95, 3 80 nanometer ratio images of four helo cells expressing ratio metric peram in the mitochondria treated with 10 micromolar A TP, and the quantification of changes in mitochondrial calcium levels of those previous four cells is shown here in this image of a hela cell expressing ratio metric peram in the mitochondria.
Heterogeneity of the calcium response in individual mitochondria as well as significant fragmentation of mitochondria is evident by 60 minutes of 0.5 micromolar stor sporin treatment. Some mitochondria have oscillatory increases in calcium shown with the white arrow, whereas others do not show significant changes in calcium level shown with a yellow arrow. This graph reveals the change in global mitochondrial calcium levels in a single helo cell after induction of apoptosis for 120 minutes with 0.5 micromolar stor sporin.
After watching this video, you should have a good understanding of how to measure dynamic changes in mitochondrial calcium levels in response to various tli using calcium medicated protein R geometric peram, which is selectively targeted to the mitochondrial matrix.
