Methoden voor-Cell bevestigd Capaciteitsmetingen in Muis bijnier Chromaffinecellen Cell

The overall goal of this procedure is to detect single vesicle, cytosis and endocytosis. This is accomplished by first obtaining a clean and complete digestion of mouse adrenal gland tissue. The second step is to dissociate and plate cells from the adrenal glands.

Next chromin cells are identified and patch clamped to obtain cell attached capacitance recordings. Ultimately, single vesicle, cytosis and endocytosis can be observed. The main advantage of this technique over existing methods such as cell attached recordings of single ion channels, is that we're able to detect exocytosis and endocytosis of single vesicles.

Visual demonstration of this method is useful as obtaining a giga seal is difficult to learn. Also, it can be difficult to identify cells amongst mixed cell types found within the plate. To prepare for the surgery begin by autoclaving a pair of large and small dissection, scissors, and two pairs of forceps.

Then place the dissecting tray on an ice bucket. Also begin to bubble the enzyme solution with 5%carbon dioxide and 95%oxygen. The solution is bright pink prior to bubbling.Next.

After euthanizing and decapitating a newborn to three day old mouse, utilize the smaller set of scissors to cut down the back of the pup following the spine from the cervical to cordal region. Be sure to cut superficially under the skin to avoid piercing the body cavity. Next, peel the skin away from the spine so that the musculature is exposed and there is a clear view of the spinal column.

Now make a trans sectional cut at the cervical spinal cord and then make two parallel cuts along the sides of the spine with one pair of forceps holding the main body of the animal. Use the other pair of forceps to grab the spinal column and peel back the spine until the kidneys are exposed. After locating the adrenal glands on top of the kidneys, use forceps to remove them.

Then carefully place the adrenal glands into a conical tube filled with lock solution. Use a second pair of forceps to dislodge the adrenal glands. If they stick to the first pair of forceps, place the conical tube on ice.

The glands can be kept in lock solution for up to two hours. At this point, check the enzyme solution to ensure that the equilibrated solution has turned from a bright pink color into a pinkish orange color. Next, add pape to the freshly bubbled enzyme solution at a final concentration of 20 to 25 units per milliliter.

Then add the propane enzyme solution to a new conical tube and use forceps to transfer the adrenal glands into the propane enzyme tube. Incubate the glands for 40 to 60 minutes at 37 degrees Celsius. Then add 75%of inactivation solution to each tube and incubate at 37 degrees Celsius for another 10 minutes.

After 10 minutes, gently transfer the glands into a newly labeled tube and wash the glands three times with culture media. After washing, place the glands into 200 microliters of the culture media, and then gently titrate the glands up and down through the pipette tip of a 200 microliter pipette. When titrating, maintain the glands at the bottom of the tube with the downward force from the 200 microliter pipette tip and engage the tissue gently and slowly Be certain to use slow, continuous strokes, never pipetting quickly or abruptly following titration, add an additional 250 microliters of culture media to bring the total volume of the culture media up to 450 microliters.

Next place, seven 12 millimeter PDL pre-coated cover slips in a 60 by 50 millimeter culture dish plate. Then plate 60 microliters of the cell containing solution onto each of the seven cover slips. Once plated, place the dish in the incubator at 37 degrees Celsius for 30 minutes.

After incubation, gently add five milliliters of prewarm culture media to the culture dish, then incubate for 24 hours prior to performing cell attached recordings. After the incubation place, the 12 millimeter cover slip containing the adrenal cells into the extracellular solution. Since the whole adrenal glands were used, the next step is to differentiate between the chromin cells and cortical cells.

In culture, the chromin cells are typically very bright with a brownish beige coloring and a near perfect circular form. In contrast, the cortical cells are smaller and dimmer than the CHRO cells. After pulling the patch pipettes as described in the text protocol, dip the pipette tip into melted wax to reduce the capacitance of the glass.

Then fire polish the tip to blow away this wax from inside the pipette tip. Next, build the pipette with the patch pipette solution so that its resistance is approximately two mega ohms. Attach the patch pipette to its manipulator and bring it within several microns of the cell.

Continue to advance the pipette looking for any slight cell deformation. Once the deformation is spotted, apply gentle suction until a giga om seal is achieved. Yielding a cell attached configuration.

Once a giga om seal is revealed, make cell attached recordings through the use of an EPC seven plus patch clamp amplifier, and a two-phase analog lock-in amplifier as described in the text protocol, set the phase of the lock-in amplifier, such that transient capacitance changes produced by gentle suction. Pulses appear only in the patch capacitance, or Im trace with no projection into the patch, conductance or retrace the patching process serves as mechanical stimulation. Since action current can be typically recorded from most cells, identify typical capacitance changes by the marked downward step.

The final step is to save the data for future analysis. Shown here is a typical cell attached capacitance recording with multiple downward steps. Each downward step is associated with a single iCal endocytosis.

Once mastered, the cell culture can be completed within two to three hours, and recordings can be performed within the following four days after its development. This technique paved the way for researchers in the field of synaptic transmission to explore synaptic vesicle recycling in neuroendocrine chromin cells.