The overall goal of the following experiment is to visualize calcium dynamics directly in the lumen of the endoplasmic reticulum of living cells. This is achieved by transducing the cells with the targeted esterase induced dye or TED vector construct to overexpress carbox esterase CS two in the endoplasmic reticulum. Next, the cells are incubated with the aceto methyl ester calcium indicator flu oh five NA, which diffuses through the plasma and ER membranes.
Then the carboxyl esterase cleaves off. The ester group releases the calcium sensitive indicator in the er. Lumen and fluorescent calcium complexes are formed.
Results are obtained that show changes in free calcium concentration in the ER based on live cell imaging. This method can help answer key questions regarding the regulation of cellular calcium homeostasis. Calcium signals are most frequently monitored in the cytosol, therefore it cannot easily be distinguished where the calcium is flowing into the cytosol from the extracellular space or from the intracellular ER calcium store.
To overcome this limitation, we have developed targeted esterase induced downloading. With this method, calcium released from the ER is monitored directly. The approach is non-disruptive.
The plasma membrane remains intact and intracellular signaling cascades are preserved. Generally, individuals n tous method will struggle because they need experience. In TED Vector expression, we have developed vector constructs expressing a cent protein fused to the Carboxylase CES two.
This allows us to easily identify cells suitable for AR calcium imaging during the experiments, which will cells that are characterized by strong red fluorescence and only moderate to strong calcium indicator fluorescence. This fluorescent labels should be evenly distributed throughout the endoplasmic reticulum and are absent in the nuclear region. Before beginning.
Prepare artificial cerebral spinal fluid or A CSF and flu oh five NAM according to the text protocol. Prewarm the A CSF to room temperature and add deep glucose aerate the A CSF with carbogen for five minutes. Then add one molar stock solutions of calcium chloride and magnesium chloride to final concentrations of two millimolar each.
After starting the live imaging microscope and computer program, begin the profusion on a dummy imaging chamber and equilibrate the tube system prewarm the inline solution heater and the imaging chamber on the microscope stage and equilibrate the system to 32 to 37 degrees Celsius. Before starting the downloading procedure to load dye into cells, add 100 microliters of pre warmed A CSF to one 0.5 microliter aliquot of five millimolar flu oh five NAM, and solubilize the solution in a water bath sonicate for 90 seconds transfer 400 microliters of prewarm imaging solution to one well of a four or 24 well plate. Add the D solution to the same well to produce 500 microliters of a five micromolar solution.
Next, carefully place a cover slip with cells in the well incubate for an appropriate period of time In a cell culture incubator at 37 degrees Celsius DI loading time and D concentration depend on the cell type cell density and experiments specific needs. Long incubation times may harm the cells, especially primary nuance and glial cells, and this is critical for the responsiveness to physiological stimuli. Incubation of more than 30 minutes will only be useful for ER calcium localization experiments to investigate the distribution of ER calcium in small micro domains, for example, MUEs of spines Immediately after the incubation transfer the cover slip to another cell culture well containing imaging solution.
To prepare for mounting the cells in the imaging chamber, use a paint brush to apply high vacuum grease to the imaging chamber. We use self-made imaging chambers for 10 to 12 millimeter cover slips with a small volume, thus allowing high perfusion speeds of up to 15 fold buffer exchange per minute. Pick up the cover slip with the flu oh five and loaded cells and add a small drop of imaging solution to keep it wet.
Then turn the cover slip upside down and mount the cells into the imaging chamber. Use a cotton swab to press the cover slip into the silicone glue and to wipe away residual buffer from the bottom of the glass cover slip. Next, exchange the dummy imaging chamber with the experimental imaging chamber.
Wash the cells for five to 10 minutes by continuous perfusion for cell selection. Use a minimal amount of laser illumination to prevent rapid photo bleaching of flu oh five and calcium two in the er, a high scan rate of two to four hertz, a small image size and a high gain set up at the microscope. According to the plant experiment.
At the beginning of the experiment, document the cellular distribution of the flu oh five and calcium label and the red fluorescent label of the red carbox cel esterase construct. In your cell of interest here, representative neurons by high resolution X, YZ image stacks perform XYT imaging to monitor ER calcium dynamics under the specific experimental conditions. Typical settings for our system are listed in table two of the text protocol Monitor changes in ER calcium under continuous perfusion with imaging solution buffer exchange rates are 1.5 milliliter per minute for cell washing and store depletion by blocking the sarco endoplasmic reticulum calcium two a TPAs and three milliliters per minute for stimulation by a TP and DHPG and agonist for the metabotropic glutamate receptor.
Here glial cells are stimulated with 200 micromolar A TP for 10 seconds. To process the images, open the image stack in the Image J program for multicolor imaging. Split the RBG channels when asked by the software.
Identify the regions of interest. Depending on the purpose, draw regions of interest around either the complete ER or small regions of the er. Use the time series analyzer plugin to read out the average pixel intensity in a region of interest.
Calculate the background corrected relative changes in fluorescence by delta F over F zero using this formula. Present relative changes in fluorescence as a trace with delta F over F zero for the Y axis and time for the x axis. In this figure, he A cells cortical astrocytes and hippocampal neurons express a red fluorescent TED vector and are labeled with the low affinity calcium indicator.
Flu oh five N.The fluorescent calcium indicator complex is localized to the endoplasmic reticulum of all cell types. This figure shows flu oh five N labeled glia cells. During CIN treatment, a bright cytosolic labeling is seen if a cell's plasma membrane is damaged both before and after CIN treatment.
This reveals that flu oh five N is also released in the cytosol by endogenous esterases. In this experiment after TED labeling with flu oh five N er calcium depletion of cultured hippocampal neurons is caused by the circa blocker cyclonic acid. Note, the enormous change in mean fluorescence density per region of interest that is observed shown here is a time lapse series with mouse cortico GL cells that are stimulated with a TP.The fluorescence abruptly drops and recovers representing ER calcium release and refill Simultaneously, the fluorescence of the red fluorescent protein decreases marginally as a consequence of slate bleaching diagrams showing the change in fluorescence intensity over time in some, but not all experiments.
The fluorescence values do not come back to the initial fluorescence intensity levels because some flu oh five N complexes are lost during stimulation. This movie shows a carboxylase two infected BHK 21 cell at high resolution labeled by flu oh five N calcium complexes and stimulated with a TP.Note that a TP induced calcium release and ER calcium store refilling is visualized in fine structures of the ER tubules. While attending this procedure, it is important to remember that only perfectly healthy cells show physiological calcium responses.
We therefore recommend to perform a series of interrelated calcium homeostasis experiments in one day with the same cell batch. Based on our experience, the dye flu of five N works best, but this calcium indicator is not very breach resistant. Therefore, this method will benefit from development of more bleach resistant low affinity calcium indicators.
After watching this video, you should have a good understanding of how to image E calcium transient with synthetic low affinity calcium indicators. These indicators enable you to monitor e calcium dynamics with high temporal resolution. For this reason, our method may be beneficial for the investigation of calcium signaling cascades involved in synaptic plasticity and to monitor changes in calcium homeostasis in cellular models for neurodegenerative diseases.
