The overall goal of this procedure is to generate rap primary interm medullary collecting duct cells or IMCD cells suitable for studying a VP mediated water reabsorption. This is accomplished by first removing the rat kidneys. The second step is to isolate the inner medulla from the surrounding outer medulla and cortex.
Next, the inner meli are digested enzymatically to release individual cells. The final step is to suspend and select for principal cells and then seed them into culture dishes. Ultimately, six to eight days after seeding the cells are ready to be used in experiments.
The cell culture system is suitable for elucidation of molecular mechanisms underlying a VP mediated water reabsorption, and possibly other CAP dependent TIC processes. In addition, the system can be used for the discovery of new drug candidates and drug targets for the treatment of diseases associated with dysregulation of a VP mediated water reabsorption. The procedure will be demonstrated by a PhD student from our lab and by Beata Eman and Andrea Giller are two technicians from our lab.
Following the timeline scene here, coat the culture dishes with collagen. Type four, the day before the procedure the following day, prepare the medium and enzyme solutions First. Add supplements to freshly prepared DMEM medium.
To prepare the enzyme solution, add one milligram per milliliter hyaluronidase, and 2.2 milligrams per milliliter. Collagenase to DPBS filter. Sterilize the solution and store it in a 50 milliliter Falcon tube.
Prepare one milliliter of enzyme solution for every two kidney inner meli digested. The most difficult step of this protocol is the dissection of the inner meela To ensure success, blood is drained from the animals before dissection. Making the shimmery border between inner and auto model easier to detect After euthanasia.
Remove the kidneys and store them in the sterile DPBS prepared earlier. Working under sterile conditions. Transfer the kidneys from the DPBS solution, supplemented with Gentamicin Nystatin onto a sterile compress to remove any excess liquid.
Next, with the sterile scalpel, remove the fatty kidney capsule while still fixed on the compress. Make a cut near the longitudinal axis of the outer or cortical side. Make sure not to cut through completely, but to leave it in one piece still connected in the renal pelvis.
Next, identify the shimmery rose colored outer medulla and the whitish inner medulla. With curved scissors, carefully dissect the inner medulla away from the surrounding outer medulla. Place the tissue in the DPBS solution prepared earlier.
Once all Meli are isolated and collected on ice, reduce the volume of DPBS solution, supplemented with Gentamicin and Nystatin to a level where they're just covered with buffer. Use a sterile razor blade to cut the Meli into cubes of approximately five millimeters cubed. Next melt and round the sharp end of a sterile pasti pipette by holding it briefly into a flame.
Once cooled, use this pipette to transfer the tissue suspension to a 50 milliliter Falcon tube containing the fresh sterile enzyme solution. Prepared earlier. Place the tube in a water bath and incubate at 37 degrees Celsius under continuous agitation for one and a half to two hours.
After this time, pipette the mixture up and down several times with the round past your pipette until the suspension becomes homogeneously turbid. Once thoroughly homogenized, centrifuge the suspension for five minutes. Next, discard the supernatant res.
Suspend the pellet and centrifuge again. Repeat this washing step once more. Re suspend the cells in fully supplemented medium and seed them into culture dishes or microtiter well plates directly after seeding isolated IMCD cells as well as erythrocytes and cell debris can be evaluated under the microscope.
This will provide an early idea of the number of cells isolated after 24 hours. Wash the cells with 600 milli osmoles, DMEM and add fresh medium. During the next week, the cells should be washed two to three times.
The IMCD cells should be ready for experiments six to eight days after seeding when grown to confluence as can be seen here. Remember to incubate the cells in medium without DBC MP and INE for 24 hours before starting the experiment. Otherwise, aquaporin two will be located mainly in the plasma membrane.
The successful cultivation of primary rat IMCD cells will result in a confluent monolayer six to eight days after seeding when stimulated with arginine, vasopressin, or forskolin aqp two translocates from intracellular vesicles to the plasma membrane as seen here. In addition, AQP two expression in IMCD cells increases upon a 30 minute challenge with a VP or forline as shown in this western blot. Following this procedure, D-N-A-R-N-A, isolation R-T-P-C-R Immunoprecipitation experiments.
Western blood analysis and immunofluorescence microscopy can be performed in order to analyze genes and gene products of inod collecting duct cells. This cell culture system, which expresses endogenous proteins is particularly attractive for use in unbiased screening approaches.
