The overall goal of the following experiment is to isolate lax Z positive cells from the neonatal mouse cochlea using flow cytometry. This is achieved by first isolating a large number of mouse cochlea in a relatively short timeframe to keep the cells viable and to have adequate starting tissue. As a second step, the LAX Z positive cells are dissociated and then labeled to prepare the tissue for sorting.
The cells are then sorted by flow cytometry. The resulting LAX Z high and lax low populations can then be used for further experimentation. This method can help answer key questions in the ear research field, such as what are the gene profiles or specific cell populations within the inner ear.
Demonstrating the procedure will be Taha Gen A medical student from our laboratory. First, fill 35 millimeter Petri dishes with HBSS and place them on an ice block. Then after using iris scissors to decapitate 12 to 15, euthanized postnatal, zero to three day old axon, two laxy reporter and wild type mice.
Place the heads onto a 60 millimeter Petri dish on a ice block. Next, use number five Dumont forceps and iris scissors to excise the mandible and tongue from the heads, and then cut through the skull base at the level of the palette it. Now remove the overlying skin of the calver and transect the skull base by inserting the scissors at the inferior part of the foramen magnum to the middle of the palate.
Make another incision with the scissors from the cut edge of the palate to the superior aspect of the calver on both sides. Being careful not to damage the otic capsule. Then complete the isolation of the temporal bone by making 2 45 degree angled incisions, starting from the superior aspect of the frame and magnum extending to the coronal cut at the hard palate.
Then place the isolated temporal bones into the 35 millimeter Petri dishes containing HBSS on the ice block and transfer the ice block to a sterile dissection hood. Next, fill another 35 millimeter Petri dish this time with PBS and place it onto the ice block. Using two number 55 forceps.
Begin the microdissection by gently removing the cortex, brainstem and cerebellum from the temporal bone overlying the otic capsule. Then without removing the otic capsule, chip away parts of the cochlear capsule beginning at the entrance of the vestibular cochlear nerve and continuing into the otic capsule. Following the exposure of the base of the cochlea, gently pull out the organ of corti and the surrounding tissues.
Then beginning at the base, remove any remaining spiral ganglion and stria ssis and transfer the cochlea to the Petri dish filled with PBS first pipette. 50 microliters of sterile PBS into the center of one to two wells of a non coated six well culture dish. Then transfer no more than 20 axin, two laxy cochlea into each droplet of PBS.
Next prewarm 50 microliter aliquots of 0.125%trypsin for about two minutes in a 37 degree Celsius water bath. And then add the trypsin to the cochlea. Taking care not to shake the dish.
Incubate the cochlea at 37 degrees Celsius for eight minutes. Terminate the trypsin ization by adding 50 microliters of soybean tripsin inhibitor to each droplet, and then add an additional 50 microliters of full media after setting a P 200 pipette man to 125 microliters. Use a 300 microliter blunt pipette tip to gently tate the cells 80 times being careful not to create bubbles.
Then after each droplet has been triturated, add an additional 200 microliters of full media to each. Well now pass the cells through a 40 micron cell strainer into a new well. Within the same six well plate and then transfer the cells from each well to individual standard fax tubes.
Next, add 100 microliters of wild type cells into each of three tubes, followed by dilution with 300 microliters of full media for the CUG only. Control dilute 10 microliters of axon, two LAX Z cells in 390 microliters of full media. Transfer the remaining axon two lax Z cells into fax tubes in 400 microliter OTs.
Then after incubating the tubes in a 37 degrees Celsius humidified incubator for 10 minutes, add 200 microliters of diluted CUG to each appropriate fax tube. Next, protecting all the tubes from light, incubate the cells in a 37 degree Celsius humidified incubator for 25 minutes. For the staining while the cells are incubating, place 10 milliliters of freshly prepared heaps plus FBS in a 15 milliliter tube and place the tube on ice.
Then add 1.8 milliliters of the ice cold heap solution to each of the fax tubes under the sterile hood with the lights off to minimize light exposure. Next, centrifuge the cells at 1200 RPM for five minutes at room temperature. Then remove the supernatant and resuspend the pellets in 300 microliters of full media.
Pool all the aliquots for the sorting sample, and then add propidium iodide to each appropriate tube to a final concentration of one microgram per milliliter. Finally, after covering the cells, once again, place them on ice. Set up the sorting gates by first collecting 1000 events of the unstained wild type cells to determine the cell distribution on the forward versus side scatter plot.
Adjust the forward and side scatter parameters as necessary. Then compensate with the unstained wild type PI stained wild type and CUG stained axon. Two LAX Z cells.
Fill two fax tubes with 500 microliters of full media. Then start the analysis with the wild type PI stained and axon two Lae CUG stained cells gate to exclude debris based on the forward versus side scatter plot. Exclude doublets and clumps using the forward scatter height versus forward scatter area Plot.
Then exclude PI positive dead cells based on the forward scatter area versus PE sci five area plot. For each gait placement, set up a plot of forward scatter area versus cascade or Pacific blue area. Analyze 10, 000 events from the sort sample tube containing axon two laxy PI and CUG stained cells using all the gates set with the wild type cells using plots from the wild type cells.
Create gates for cascade blue high cells that are less than 1%of the total number of events. The cascade Blue High Gates should now contain about 15 to 20%of the cells in the Axon two laxy sample. Making these axon two high cells using the Axon two laxy cell analysis plot now create a gate for the least flu, 15 to 20%of cells using the aforementioned gating strategy.
Sort the cells into the fax tubes containing full media, keeping the collection tubes in a chamber cooled to four degrees Celsius during the sort. Record the number of cells counted by the flow cytometer for each population in these two figures. The side scatter areas versus the forward scatter areas of the dissociated wild type on the left and axon two LA Z on the right.
Cochlear cells are shown. Note the gating of the distinct groups of events of a much larger size than those events closer to the Y axis designated as the debris negative population. Typically 40 to 45%debris free cells are recovered from both wild type and axin two LAX Z cochlea in these two panels.
The forward scatter height versus the forward scatter area is used to exclude doublets from the previously gated debris. Negative population events deviating from the expected linear nature of the plot are excluded. This preparation typically yields 71.1%events within the designated gate.Here.
Propidium iodide as detected by the PE SCI five channel, is used to exclude dead cells as observed in these scatterplots. About 95%of the sorted cells were viable and did not take up PI PI in these panels. The Cascade blue channel was used to detect the fluorescent substrate CUG as seen in the figure on the right.
On average, the top roughly 20%CUG positive cells are lae high cells that correlate to about 15, 000 axon, two high cells per 10 to 12 animals. After watching this video, you should have a good understanding of how to prepare and isolate laxy high cells using flow cytometry from inner ear tissues for both cell Turing experiments and experiments such as gene profiling.
