The overall goal of this imaging technique is to monitor fluctuations in lumenal SR calcium levels in response to calcium mobilizing agents specifically in vascular smooth muscle cells. This method can be helpful in elucidating cellular calcium cycling in a more direct fashion. For example, it's very important to know the relation with between our calcium transport in the sarcoplasmic reticulum and ER stress.
The advantage of this technique is that we can directly measure the calcium inside the lumen of the sarcoplasmic reticulum rather than to have to extrapolate it from indirect measurements of changes in the cytoplasm. Demonstrating this technique will be Gabriela Ziomek who is a graduate student in my laboratory. To begin this procedure, prepare a one to 25 dilution of protein mixture in DMEM.
Next, transfer 200 microliters of protein mixture DMEM to each 35 milometer glass-bottomed plate to fully cover the bottom inner glass circle. Leave the plates at room temperature inside the safety cabinet for at least 30 minutes. In the meantime, warm DMEM with 10 percent NCS to 37 degrees Celsius in a water bath.
Afterward, remove the DMEM from the culture flask using a glass pipette. Subsequently, wash the flask bottom with warm sterile PBS to remove the remaining DMEM residue. To dislodge the cultured smooth muscle cells from the flask bottom, wash the flask quickly with one milliliter of trypsin and remove it.
Then add in another milliliter of trypsin. Swish the flask for about 30 to 60 seconds to ensure that the enzyme makes contact with the entire bottom of the flask before removing it. Once the cells have been dislodged with trypsin, add fresh DMEM with 10 percent NCS to the flask to stop the trypsin reaction.
Transfer the resulting cell solution from the flask to a clean labeled 50 milliliter conical tube. Then add one milliliter of fresh DMEM plus 10 percent NCS to each plate being prepared. After that, gently invert the tube containing the cell solution multiple times to break up any pellet that may have formed.
Pipette the desired volume of gently mixed cell solution into each plate. Subsequently, swirl each plate thoroughly clockwise or counter clockwise to ensure equal distribution of cells across the glass at the bottom. It's necessary to add enough cells to the plate to make sure that they reach high enough confluency over the next 48 hours for effective imaging.
Following the addition of medium and cell solution to each plate, incubate the plates at 37 degrees Celsius with five percent carbon dioxide for at least one hour to allow the cells to attach properly to the glass bottom. Check the plates under a microscope at fifteen minute intervals until the cells are clearly attached. Then retrieve the Adenavirus D one SR aliquat from minus 80 degrees Celsius storage and transport it to the biological safety cabinet in a mobile ice bath.Afterward.
Pipette the desired dose of chilled D one SR to each plate and incubate the infected cells at 37 degrees Celsius overnight. The next day, replenish the cells with one milliliter of fresh DMEM plus ten percent NCS. Subsequently, incubate the plates in a humidified incubator at 37 degrees Celsius with five percent carbon dioxide overnight.
Following overnight incubation, remove the culture medium from the plate. Wash the culture plate with one milliliter of warm physiological HEPES-PSS buffer three times. Next, add one milliliter of fresh warm HEPES-PSS buffer immediately prior to recording.
Perform initial imaging using the FRET SE application by opening the confocal microscope's associated software. Once the application is open, click the setup tab to adjust the desired experimental settings. Manually change the channel settings so that the cells are excited in sequence at 440 nanometers for donor and FRET and 513 nanometers for acceptor.
Then set the emission wavelengths at 488 nanometers for donor and 535 nanometers for FRET and acceptor. Subsequently, set the light intensity at 15 percent transmission with 150 millisecond excitation exposure time of cells and 10 second intervals between exposures. Next stabilize the plate on the microscope stage.
Use the live button to check the image resolution. Adjust the settings further for the desired resolution. Under the setup tab, take an image of the sample using the capture image button.
Now moving to the evaluation tab, choose the appropriate method to calculate the FRET efficiencies for the experiments being preformed. Method three which uses ratiometric calculation is recommended for experiments involving Cameleons such as the D one SR indicator. After that, switch to the graph tab in order to observe the intensity values being recorded in a graph.
If desired, draw an ROI in the image viewer to observe the corresponding average intensity of this area of interest on the graph. Start recording to collect the data for at least three minutes to ensure a steady base line signal prior to introducing the agent of interest. Then, introduce a calcium moving agent as a tool for depleting SR calcium by applying it directly into the plate near the region of interest being recorded.
Continue the recording for a desired duration after treatment. Afterward, capture the ratiometric FRET serial images with a confocal inverted microscope using the FRET SE application. In this step, to prepare the culture plates of rat aortic smooth muscle cells wash the culture plate with one milliliter of warm physiological HEPES-PSS buffer three times.
Then, mix the indicator with one milliliter of warm HEPES-PSS and load it onto the culture plates. Incubate the smooth muscle cells with the indicator for one hour at room temperature. After an hour, remove the indicator from the culture plate and wash it with one milliliter of warm physiological HEPES-PSS buffer three times.
Then, add one milliliter of fresh warm HEPES-PSS buffer immediately prior to recording. Capture serial images with the confocal inverted microscope. Subsequently, introduce the calcium moving agent as a tool for depleting SR calcium and continue recording for the desired amount of time post treatment.
Shown here are the representative snapshots of a smooth muscle cell transfected with D one SR indicator using CFP, FRET, and YFP band pass filters. This figure shows the real time pseudo-color snapshots of the cultured rat aortic smooth muscle cells transfected with D one SR.Treatment with two micromolar of Thapsigargin showed a decrease in signal intensity which indicated a significant drop in SR calcium content due to Thapsigargin induced calcium release. And this graph shows the representative trace for the normalized D one SR ratio in the cultured smooth muscle cells treated with two micromolar Thapsigargin which confirmed a significant drop in SR calcium.
Here are the representative snapshots of the cultured rat aortic smooth muscle cells loaded with cytoplasmic calcium indicator and treated with two micromolar Thapsigargin. As shown, Thapsigargin causes a significant increase in signal intensity within the cytoplasm due to the release of calcium from the SR.And this graph shows a representative trace for the normalized Fluo four AM ratio in the cultured smooth muscle cells treated with two micromolar Thapsigargin. The depicted increase in calcium signal is considered to be proportional to the amount of calcium released from the SR.Once mastered, this technique can be done in approximately 48 hours from start to finish if it is done properly.
While attempting this procedure, it's important to keep the cells in the described conditions to ensure their health prior to transfection with the calcium indicator. After watching this video, you should have a clear understanding of how to prepare cultured cells to allow for measurement of lumenal SR calcium levels using the D one SR indicator.
