This protocol describes a technique to study mechanically-evoked calcium event in an ex vivo urothelial preparation. The technique is relatively easy to execute and it could be adapted to study mechanically-evoked calcium event in other native tissue preparations. Fabricate the micropipettes by placing the capillary glass in the puller and adjusting it with the capillary-retaining knobs.
Pull the glass capillary in two steps as per the manufacturer's instructions. Close the micropipettes'tip using a microforge with the heater-adjustment knob set at 60. After exposing the bladder and the pelvic bone of the euthanized mouse, crack the pelvic bone to expose the urethra and cannulate the urethra with a 24-gauge catheter.
Use a 6-0 suture to secure the catheter to the urethra. Harvest the bladder and urethra and affix them to a silicone rubber holder pad bathed in the recording solution. Separate the bladder mucosa from the underlying muscular layer with fine forceps.
Cut open the bladder mucosa and pin it down with the urothelium facing up to a silicone elastomer insert in the bottom of a 35 millimeter diameter tissue culture dish. Mount the tissue culture dish with the pinned bladder mucosa in the microscope stage equipped with a culture dish incubator with resistive heating elements. Perfuse the cell culture dish continuously at a rate of 1.7 milliliter per minute with a recording solution warmed at 37 degrees Celsius with an inline heater.
Maintain the temperature of the tissue dish incubator and solutions at approximately 37 degrees Celsius with a dual channel bipolar temperature controller. To record mechanically-induced calcium ion transients, submerge the micropipette in the recording solution. Under bright-field illumination, using a low magnification scanning objective, move the micropipette to the center of the field.
Move the micromanipulator in the vertical plane and adjust the focus to move the pipette near the surface of the urothelial tissue. Switch to the objective with a higher magnification and numerical aperture suitable for immunofluorescence. Set the micromanipulator to fine and move the pipette near the top of the target cell.
If necessary, adjust the focus and the position of the pipette. Open the stimulation protocol in the electrophysiology software and set it to play. Adjust the focus and press start in the experiment manager to initiate data acquisition.
This drives the piezoelectric actuator and generates a file with the images of the experiment. When the cell is stimulated, an increase in fluorescence is observed. To quantify the fluorescent intensity, open the image file in the imaging software and select the count and measure window.
Play the movie to identify the stimulated cell. Select the polygon tool and draw an ROI on the boundaries of the cell that was poked. Go to the measure window, select intensity profile, set the measurement to overtime, results to average, background subtraction to none, and then press execute.
The mean fluorescence intensity will be computed over time. To export the intensity profile data, click on the Excel icon in the intensity profile results window and perform data analysis using scientific graphing and data analysis software. In this study, the umbrella cell was poked to assess mechanically-evoked calcium transients.
The image captured at 10 seconds shows a fluorescent view of the stimulating pipette positioned on top of an umbrella cell expressing a fluorescent calcium sensor GCaMP5G. Mechanical stimulation of the umbrella cell expressing calcium sensor causes a deformation followed by a rapid increase in fluorescence emission. The graph demonstrates a change in the fluorescence intensity plotted as a function of time for several cells.
Poking evokes a calcium response in most umbrella cells transduced with a fluorescent calcium sensor. The most important step is harvesting the bladder and urethra, followed by affixing them to a silicone rubber holder pad. Avoid tissue damage when separating the bladder mucosa from the underlying muscular layer.
Imaging methods such as the one described here can provide unique insight into how mechano-activated channels sends changes in their native environment and generate physiological responses.
