To begin, pull borosilicate glass capillaries with an outer diameter of two millimeters and an inner diameter of 1.56 millimeters, following appropriate settings. Prior to filling the capillary before each experiment, use fine tweezers to break open the tip of the capillary. For loading the capillary, use the stimulating solution at three to five times the normal concentration of the stimulant required.
With a thin plastic transfer pipette, slowly inject the stimulating solution into the capillary until 3/4 of its length is filled. Eliminate any air bubbles that may form during the filling process by gently flicking the capillary. Then, mount the filled capillary on the micromanipulator.
Configure the single cell delivery system settings to provide a 10-second pulse at five psi. Connect the borosilicate capillary mounted on the micromanipulator to the single cell delivery system pipette holder. Apply a brief puff lasting two seconds at a pressure of 20 psi, while observing the capillary tip to confirm that the solution is being dispensed through the tip's end.
The setup is now ready for use with microscopy for real-time imaging of a single mouse sperm.
