The overall goal of this imaging approach is to monitor the nitric oxide signals in real time on the level of individual cells using a novel class of genetically encoded fluorescent nitric oxide probes, the geNOps. Considering the importance of nitric oxide in biology and physiology, a visualization of this molecule in cellular or even subcellular level has been desired since it was discovered. We visualize the nitric oxide signals with novel, rather simple built, fluorescent protein-based probes, the geNOps.
The geNOps respond directly and immediately to nitric oxide by fluorescence quench. This means you can monitor the real-time kinetics of the generation, diffusion, and degradation of nitric oxide in single cells. The main advantage of geNOps is that you actually can look at nitric oxide signals with high spatial and temporal resolution using a conventional fluorescence microscope.
Begin this procedure by replacing the storage buffer of the endothelial cell line or HEK293 cells with one milliliter per well of the iron(II)booster solution at room temperature. Incubate the cells for exactly 20 minutes in darkness. The iron loading procedure is essential to get full responsiveness of the probes and might be adapted for different cell types and applications.
Afterward, wash the cells once with storage buffer, and incubate each well with two milliliters of storage buffer for at least two hours at room temperature in order to allow the cells to equilibrate. Then, replace the storage buffer with 3.3-micromolar Fura-2AM in one milliliter of storage buffer. Protect the cells from light, and incubate them for 45 minutes.
After 45 minutes, wash the cells twice with storage buffer, and once again incubate them for at least 30 minutes in order to allow the cells to equilibrate. To begin the procedure, first fix a 30-millimeter coverslip coated with the endothelial cell line or HEK293 cells in a metal perfusion chamber. Switch on the imaging system, and then place the metal perfusion chamber on the microscope.
Connect the influx tube to the buffer reservoirs, and then turn on the perfusion system. Now connect the efflux to a vacuum pump, ensuring a consistent flow and avoiding draining of the coverslip. Start the gravity-driven perfusion with physiological calcium buffer using a semi-automatic perfusion system.
Then, define the imaging settings using the respective software. Select the excitation wavelengths 340 nanometers and 380 nanometers for Fura-2 imaging and 480 nanometers for exciting G-geNOp. Next, select the imaging region by moving the xyz-table of the microscope until several fluorescent cells are in focus.
Then, define the regions of interest. Draw the regions covering several whole single fluorescent cells for each imaging field. Following that, define a background region of similar size.
Start collecting data on an inverted and advanced fluorescent microscope with a motorized sample stage and a monochromatic light source. Alternatively, excite the cells at 340 nanometers and 380 nanometers for Fura-2AM and 480 nanometers for G-geNOp, respectively. Set respective exposure times so that a clear fluorescence signal is detectable over time for all channels.
Collect the emitted light at 510 nanometers for Fura-2AM, 520 nanometers for G-geNOp using a CCD camera with appropriate filter set consisting of the 500-nanometer exciter, a 495-nanometer dichroic, and a 510-520-nanometer emitter. Then, record one total frame every three seconds. Record the first three minutes in physiological calcium buffer to obtain the baseline of respective fluorescence signals.
Once a stable baseline fluorescence is observed, switch to 100-micromolar histamine or ATP containing physiological calcium buffer to stimulate the cells for three minutes. Subsequently, switch back to the physiological calcium buffer without histamine or ATP and L-NNA for five minutes to remove the compounds from the cells. Afterward, administer 10-micromolar NOC-7 in the physiological calcium buffer for two minutes using the perfusion system.
The nitric oxide donor strongly affects G-geNOp fluorescence, and it usually decreases the cells by more than 20%in response to 10-micromolar NOC-7. In EA.hy926 cells, the NOC-7 effect is stronger compared to the agonist-induced G-geNOp fluorescence quench. Following that, wash out the nitric oxide-releasing compound for approximately 10 minutes with the physiological calcium buffer, and stop recording once the basal fluorescence is recovered.
Shown here, are the representative wide-field fluorescence images of EA.hy926 cells expressing G-geNOp that are loaded with Fura-2AM. And here, is the representative time course of the simultaneous recordings of Fura-2 and G-geNOp signals in response to 100-micromolar histamine. As indicated, histamine was removed after three minutes using a perfusion system.
These curves represent the simultaneous recordings of cytosolic calcium and nitric oxide signals of a single Fura-2AM-loaded endothelial cell transiently expressing G-geNOp. The cells were stimulated with 100-micromolar histamine for three minutes in a calcium-containing buffer. And shown here, are the representative simultaneously recorded calcium and nitric oxide signals over time of an EA.hy926 cell that was pretreated with 500-micromolar L-NNA prior to measurement.
Cells were treated with 100-micromolar histamine in the presence of two-millimolar calcium. This figures shows the average curves representing cytosolic nitric oxide signals in response to one-micromolar ATP followed by a second cell stimulation with 100-micromolar ATP. Once mastered, this technique allows you to visualize nitric oxide signals in a real-time manner with minimal effort.
I believe that this technique will pave the way for researchers to explore and reinvestigate nitric oxide signals in various cell models. After watching this video, you should have a good understanding how to use the geNOps technology for real-time imaging of nitric oxide in your model system.
