My research mainly focuses on cardio cerebrovascular electrophysiology. This study describes a method for isolating primary vascular muscle cells and utilizing whole cell pinch clamp technology to assist their electrophysiological properties. Fresh cell isolation has limitations such as the impact of temperature on enzyme separation, varying digestion times for different blood vessels, and instant ability in the cells native environment.
Additionally, pinch clamp technology presents challenges in ceiling and membrane rupture which requires skilled operation. This protocol facilities research is in readily acquiring active primary cells from manual arteries and quickly must ring the application of the pinch clamp technique. To begin, fill a Petri dish with cold physiological saline solution saturated with 95%oxygen and 5%carbon dioxide.
Place an isolated rat brain into the Petri dish. Position the brain ventrally upward in the Petri dish and secure it with needles. Under a light microscope, locate the basilar artery.
With autoclave, tweezers and scissors, carefully remove the surrounding tissue. Next, insert a two centimeter long wire with a diameter of 25 micrometers into the isolated basilar artery. Gently rub the inner wall of the artery with the wire to effectively remove the vascular endothelium.
To isolate smooth muscle cells, first, preheat the solutions in test tubes one to three. Transfer the isolated basilar artery to test tube two. Incubate the mixture at 37 degrees celsius for 30 minutes while continuously injecting a mixture of 95%oxygen and 5%carbon dioxide into the tube.
Next, transfer the basilar artery from test tube two to test tube three and incubate. Transfer one milliliter of preheated cell separation solution from test tube one into test tube three. Continue injecting 95%oxygen and 5%carbon dioxide while maintaining enzymatic treatment for three minutes.
Triturate the basilar artery preparation to release cells. Next pipette four milliliters of cold separation solution to test tube three to terminate the enzymatic process. Centrifuge the mixture at 59 G for six minutes.
With a pipette, discard the supernatant. Then add cold separation solution again. After the last wash, remove the supernatant.
Leave one milliliter of the cell suspension. Store the cell suspension at four degrees celsius for six to eight hours. Transfer 100 microliters of the cell suspension to a bath.
Add one milliliter of extracellular fluid to the bath and let it rest. To begin, turn on a micropipet polar. Place a glass tube in the polar.
On the control panel select program one. Click enter and access program one. Now perform a ramp test by clicking ramp on the control panel to measure the heat value of the glass tube.
Insert a new glass tube. Then select program one, and click enter to fabricate the micro pipette. To record the care current, first launch the data acquisition software.
Select file, then click on set data file names to establish a data storage path. Enable the membrane test function in the tools menu. Place a sample of isolated smooth muscle cells from a rat's basilar artery in the center of a microscope's camera view.
Immerse the reference wire tip into the bath solution. Then fill 20%of the fabricated micro pipette with the intracellular solution. Secure the loaded micro pipette on the recording electrode holder.
Now with a syringe, apply positive pressure. Move the pipette tip into the bath solution and adjust the pipette offset on the signal amplifier software to set the current baseline to zero picoamperes. Gently press the pipette against the cell membrane.
Observe an increase in seal resistance to at least one magome. Remove positive pressure and apply negative pressure. To establish high resistance, with a seal resistance of at least one giggome, Set the holding voltage to minus 60 millivolts and compensate for electrode capacitance Using CP FAST and CP slow.
Apply brief negative pressure to rupture the cell membrane, forming a whole cell configuration. Select whole cell in the signal amplifier software. Then click auto for membrane capacitance compensation.
Load the kir protocol and begin data recording. Freshly isolated smooth muscle cells with spindle morphology were isolated. The cells were healthy and suitable for patch clamp experiments.
A typical cure current exhibited inward rectification at negative voltages with minimal or no potassium ion current flow at positive voltages. Barium chloride at concentrations of 100 to 300 micromoles per liter effectively inhibited kir channel activity confirming the current as kir. Sodium nitroprusside increased kir current indicating the role of kir channels in SNP induced vasodilation.
Comparison of current voltage relationships demonstrated a decrease in current density in the presence of barium chloride, and an increase with SNP.
