The overall goal of this procedure is to separate the different types of cells found in the testes. First, harvest a mixed population of cells from the mouse. Testes load the cells and BSA gradient into the stay put apparatus.
Now allow the cells to sediment through the BSA gradient. Then collect the fractions and combine them based on cell composition. Ultimately, velocity sedimentation with the stay put is used to separate different types of cells from the testes based on differences in size and density.
The main advantage of this technique over existing methods like facts, is that one can isolate large amounts of different cell types with fairly high purity using simple glassware and gravity. Secure the two, two liter cylinders and the cell loading chamber to the top platform and connect all with two small pieces of tubing with tube clamps. Clamp all the tubes closed and seal the spout on the rightmost two liter cylinder.
Place a small stir bar in the cell loading chamber and a larger stir bar in the leftmost two liter cylinder. Then place the two liter sedimentation chamber on the platform. Position the metal baffle directly on top of the opening in the bottom of the sedimentation chamber to prevent vortexing of the liquid and disruption of the cell gradient during fraction collection.
Now put the lid on the sedimentation chamber. Apply a very small amount of vacuum grease to the ground glass joint of the three-way stop cock. Then clamp the stop cock to the bottom of the sedimentation chamber, connecting the ground glass joints of the stop cock and the sedimentation chamber.
Next, connect the cell loading chamber to the right outlet of the stop cock with tubing. Close the stop cock, then attach the cell fractionation tubing to the left outlet of the stop cock and clamp the small tube at the very bottom. Pour the 2%BSA solution in the left two liter cylinder.
Pour the 4%BSA solution in the right two liter cylinder. Make sure there are no large bubbles in the tubing that connects these cylinders. Pour Krebs buffer into the cell loading chamber and fill the tubing connecting this chamber with the sedimentation chamber.
Verify that there are no large bubbles that might disturb the gradient. If necessary, squeeze or flick the tubing gently to remove the bubbles. Confirm that all of the crebs is in the tube and not in the cell chamber.
Allow a small amount of Krebs buffer to flow into the sedimentation chamber. Then drain almost all the buffer into the cell fractionation tubing. In order to fill the tube and remove any large bubbles, place the fraction collector directly under the sedimentation chamber.
Check that all the fraction tubes are in place and cover with plastic wrap to prevent contamination. Deencapsulate the testes in a separate plate containing eight milliliters of Krebs. Make an incision in the thin membrane that surrounds the seminiferous tubules with one pair of forceps.
Hold the membrane now with another pair of forceps. Push the tubules out of and away from the membrane. Transfer four milliliters of Krebs containing the tubules to each conical tube containing 25 milliliters of collagenase solution.
Shake at 33 degrees Celsius for 10 minutes until the tubules develop a spaghetti like appearance. Next, allow the tubules to settle for five minutes to the bottom of the tube. Pour out the supernatant and wash twice with 25 milliliters of Krebs at room temperature, allowing the tubules to settle to the bottom of the tube each time.
Leave approximately five milliliters of Krebs in each tube. After incubating the tubules with trypsin. Use a wide bore pipette to agitate the solution containing the tubules.
Pipetting them in and out approximately 10 times. The solution should begin to look more like a single cell suspension. Now filter each 30 milliliter single cell suspension through a 100 micron mesh cell strainer.
After combining the cell suspensions, count the total number of cells. Make sure the stop cock is positioned to direct the flow from the cell chamber into the sedimentation chamber. Turn both stir bar plates onto a low setting.
Then pour the cell suspension into the cell chamber. Open the stop cock for the cells to flow slowly into the sedimentation chamber at a rate of 10 milliliters per minute. Then close the stop cock to wash the cells out of the cell chamber.
Pour five milliliters of 0.5%BSA solution and drain into the sedimentation chamber at a rate of 10 milliliters per minute. Close the stopcock and repeat the BSA wash four more times. Now open the clamps between the cell chamber and the two two liters cylinders to immediately drain the liquid into the sedimentation chamber at a rate of 40 milliliters per minute, adjust the flow rate so it takes approximately 20 to 30 minutes to load the BSA gradient into the sedimentation chamber.
Monitor for a thin undisturbed layer of cells lying on top of the BSA gradient. Once most of the BSA is loaded, close the stop cock and turn it to the position that will allow liquid to drain from the sedimentation chamber into the fractionation tube. Now turn off the stir plates and allow the cells to sediment for one hour and 45 minutes.
After sedimentation is complete, attach the fraction tube to the fraction collector. Adjust the flow rate with the stop cock so that 10 milliliters per collection tube is collected every 45 seconds. Once all fractions are collected microscopically analyze the fractions for different cell populations.
Different cell types can be distinguished based on cell size and nuclear morphology to determine the purity of each fraction. Transfer 10 microliters of DPI stained cells onto a slide. Place a cover slip and analyze with a fluorescence microscope.
Pull the fractions that are similar in size and nuclear morphology to create populations of cells. A typical preparation of approximately 22 testes. With this stay put procedure yields approximately 10 to the eighth cells.
Perper metagenic cell type fractions can be analyzed quickly using a combination of light and fluorescent microscopy. Myotic, spermatogonia and somatic diploid cells are the largest cells found in the testes and will contain large nuclei that stain relatively homogenously with dpi. Round spermatids are smaller cells with smaller round nuclei, generally with a brightly staining chromo center condensing elongating spermatids are small cells that have sickle shaped nuclei.
Once cell fractions are combined, purity can be further determined by western blood analysis of the cell lysates. Common markers of myotic cells are the Synap Neal Complex one proteins. Common markers of condensing spermatids are transition proteins or protamine.
Once mastered, this technique can be done in seven hours if it's performed properly after its development. This technique paved the way for researchers in the field of spermatogenesis to explore cellular processes in different cell types from the testes.
