We thank S. Heller, K. Oshima, R. Nusse, and Y. Zeng for fruitful discussions and C. Tang, A. Lee, E. Liaw, and the Stanford Shared FACS Facility staff for technical assistance. This work was supported by Howard Hughes Medical Institute Medical Research Training Fellowship, Stanford University Medical Scholars program (both to T.A.J.), Stanford University Dean's Fellowship (to R.C.), American Otological Society, Triological Society, Percy Memorial Award, the Akiko Yamazaki and Jerry Yang Faculty Scholar Fund, and NIDCD/NIH K08 DC011043 (all to A.G.C.).

1. Microdissection of the cochlea
<ol>
	<li>Prepare dissection microscope under hood by wiping surfaces with 70% ethanol and setting out ice blocks.</li>
	<li>Prepare 10-15 35 mm Petri dishes under sterile condition and fill half-way (~2 ml) with sterile Hank's Balanced Salt Solution with Calcium (HBSS).</li>
	<li>Prepare two 35 mm Petri dishes under sterile condition and fill half-way (~2 ml) with sterile 1x phosphate buffered solution (PBS).</li>
	<li>Sterilize micro-forceps in 50 ml conical Falcon tube with 70% ethanol.</li>
	<li>In a separate room from the sterile hood, use iris scissors to decapitate 12-15 postnatal (P) 0-3-day-old Axin2LacZ/+ reporter mice and place heads onto a 60 mm Petri dish on ice block.</li>
	<li>Using #5 Dumont forceps and iris scissors, excise the mandible and tongue and cut through the skull base at the level of the palate.</li>
	<li>Remove the overlying skin of the calvaria and transect the skull base by inserting the scissors at the inferior part of the foramen magnum to the middle of the palate.</li>
	<li>Make another incision with iris scissors from the cut edge of the palate to the superior aspect of the calvaria on both sides, being careful not to damage the otic capsule.</li>
	<li>Complete isolation of the temporal bone by making two 45-degree angled incisions starting from the superior aspect of the foramen magnum extending to the coronal cut at the hard palate.</li>
	<li>Place isolated temporal bones in 35 mm Petri dishes containing HBSS on ice block.</li>
	<li>Collect temporal bones from about 12-15 animals that have been sacrificed, for a total of 24-30 temporal bones.</li>
	<li>Transfer Petri dish with isolated temporal bones to previously prepared sterile dissection hood (1.1).</li>
	<li>Transfer four temporal bones to each pre-prepared 35 mm Petri dish containing HBSS (1.2) and place on ice block.</li>
	<li>Begin microdissection using two #55 forceps to gently remove the cortex, brain stem, and cerebellum in the temporal bone overlying the otic capsule.</li>
	<li>Without removing the otic capsule, chip away from parts of the cochlear capsule beginning at the entrance of the vestibulocochlear nerve into the otic capsule.</li>
	<li>Following exposure of the base of the cochlea, gently pull out the cochlea and surrounding tissues. Remove any remaining spiral ganglion and the stria vascularis beginning at the base.</li>
	<li>Transfer cochlea to 35 mm Petri dish containing PBS on ice block.</li>
	<li>For optimal tissue viability, the total time of dissection should be less than 60 min.</li>
	<li>Repeat above steps for 3-5 wild-type (WT) P0-P3 mice as controls for Axin2-LacZ mice.</li>
</ol>
2. Cell dissociation (modified from previously described methods)2,10
<ol>
	<li>Place 50 &mu;l sterile PBS in the center of a well in a 6-well non-coated culture dish.</li>
	<li>Transfer up to 20 Axin2LacZ/+ cochleae into each 50 &mu;l droplet of PBS. A new 50 &mu;l droplet of PBS should be used for more than 20 cochleae.</li>
	<li>Pre-warm 50 &mu;l aliquots of 0.125% trypsin for ~2 minutes in a 37&deg;C water bath.</li>
	<li>Add 50 &mu;l of pre-warmed 0.125% trypsin to PBS containing the cochleae.</li>
	<li>Place 6-well dish at 37&deg;C for 8 min, taking care not to shake the dish.</li>
	<li>To terminate trypsinization, add 50 &mu;l of soybean trypsin inhibitor to each 100 &mu;l droplet.</li>
	<li>Add an additional 50 &mu;l of Full Media (FM) (IGF-1, EGF, bFGF, HS, ampicillin in DMEM/F12)2</li>
	<li>Using a 300 &mu;l blunt pipette tip with a p200 pipetteman set at 125 &mu;l, gently triturate cells 80 times, being careful not to create bubbles.</li>
	<li>Following trituration of each droplet, add an additional 200 &mu;l of FM to each well.</li>
	<li>Pass cells through a 40 &mu;m blue Falcon cell strainer into a new well.</li>
	<li>Transfer cells to standard Falcon FACS tubes (approximately 300 &mu;l remains following trituration and filtration).
	<ol class="lalpha">
		<li>Split cells from wildtype cochleae into three tubes, diluting 100 &mu;l with 300 &mu;l FM in each tube. One tube will be used as a blank control, one will be stained with propidium iodide (PI, 1 &mu;g/ml) only (not to be added at this step), while the last one will be stained with 3-carboxyumbelliferyl &beta;-D-galactopyranoside (CUG, see 2.15) to detect background fluorescence.</li>
		<li>Place 400 &mu;l of dissociated cells from Axin2-LacZ cochleae in each FACS tube. As a CUG only control, dilute 10 &mu;l of cells in 390 &mu;l FM (see tube F in Step 2.15). Bring volume of any remaining tubes to 400 &mu;l.</li>
	</ol>
	</li>
	<li>Incubate cells in a 37&deg;C humidified incubator for 10 min.</li>
	<li>Prepare CUG solution:
	<ol class="lalpha">
		<li>Prepare 200 &mu;l of CUG for each FACS tube requiring staining.</li>
		<li>Dilute CUG in FM 1:50 in a dark hood with minimum light exposure.</li>
		<li>Pre-warm diluted CUG at 37&deg;C for 3-5 minutes.</li>
	</ol>
	</li>
	<li>Next, add 200 &mu;l of diluted CUG to each appropriate FACS tube. For tubes not designated to have CUG, add FM only (see 2.15).</li>
	<li>At this juncture, there should be five categories of tubes with the following contents (do not add PI at this step):
	<ol class="ualpha">
		<li>Wildtype - without CUG or PI (for compensation, see step 3.5-6)</li>
		<li>Wildtype - with PI only (for compensation)</li>
		<li>Wildtype - with CUG and PI (for control against Axin2-LacZ cells)</li>
		<li>Axin2-LacZ - with CUG only (for compensation)</li>
		<li>Axin2-LacZ - with CUG and PI (for actual cell sorting)</li>
		<li>Axin2-LacZ - with CUG and PI (for event analysis)</li>
	</ol>
	</li>
	<li>Protect all tubes from light and incubate them in a 37&deg;C humidified incubator for 25 minutes.</li>
	<li>Prepare fresh HEPES + Fetal Bovine Serum (FBS) solution for halting reaction following incubation:
	<ol class="lalpha">
		<li>Dilute 400 &mu;l of freshly thawed FBS in 10 ml of sterile 10 mM HEPES (in PBS) in a 15 ml Falcon tube.</li>
		<li>Place tube on ice until completion of 2.16.</li>
	</ol>
	</li>
	<li>Add 1.8 ml of ice-cold HEPES solution to each FACS tube under a sterile hood with lights off to minimize light exposure.</li>
	<li>Centrifuge cells at 1200rpm for 5 minutes.</li>
	<li>Remove supernatant and re-suspend cells in 300 &mu;l FM, pool all aliquots for the sorting sample (Axin2-LacZ stained with CUG and PI).</li>
	<li>Add PI to each tube to achieve a final concentration of 1 &mu;g/ml.</li>
	<li>Place cells on ice and protect from light.</li>
</ol>
3. Flow Cytometry
<ol>
	<li>We use a BD Aria II equipped with 488-nm, 633-nm, and 407-nm lasers. Detection of CUG was done using the 407-nm violet laser and PI using the 488-nm blue laser at the Stanford Shared FACS Facility.</li>
	<li>A trained flow cytometer operator is required. Proceed to the next step after warming up the lasers, calibrating the cytometer detectors using manufacturer instructions, and setting optimal sorting parameters. In our experience, we found the use of a 100 &mu;m nozzle optimizes cell viability for subsequent experiments.</li>
	<li>The following parameters were used: threshold at 5,000 events, flow rate less than 3,000 events/second, forward scatter (FSC) at 125 volts and side scatter (SSC) at 250 volts, PE-Cy5 channel was used for PI and Cascade Blue for CUG.</li>
	<li>Flush cytometer chamber with 70% ethanol and PBS twice each in that order.</li>
	<li>Begin analysis with unstained, wildtype cells (tube A in Step 2.15). Collect 1,000 events to determine cell distribution on the FSC versus SSC plot. If scatter is not desired, then adjust FSC and SSC parameters.</li>
	<li>Perform compensation using unstained WT (tube A), PI-stained WT (tube B), and CUG-stained Axin2-LacZ (tube D) cells. Compensation will allow for more accurate signal discrimination among the different laser wavelengths.</li>
	<li>Start analysis with wildtype PI- and CUG-stained cells (tube C) and set gating parameters for:
	<ol class="lalpha">
		<li>Exclusion of debris based on FSC versus SSC plot.</li>
		<li>Exclusion of doublets and clumps using FSC-height versus FSC-area plot.</li>
		<li>Exclusion of dead cells based on FSC-area versus PE-Cy5-area plot.</li>
		<li>Set up plot of FSC-area versus Cascade Blue-area with any gate placement.</li>
	</ol>
	</li>
	<li>Next, analyze 10,000 events from sort sample tube that contains Axin2-LacZ PI and CUG stained cells (tube F).
	<ol class="lalpha">
		<li>All gates set using wildtype cells should be applied here.</li>
		<li>Using plots for the wildtype cells, create gates for Cascade Blue-high cells that is less than 1% of the total number of events.</li>
		<li>This same gates of Cascade Blue-high now encompass approximately 15-20% of cells in the Axin2-LacZ sample, making these Axin2-high cells.</li>
		<li>Using the Axin2-LacZ cells' analysis plot, create a gate for the least fluorogenic 15-20% of cells, henceforth Axin2-low cells.</li>
	</ol>
	</li>
	<li>Based on the aforementioned gating strategy, sort cells into FACS tubes containing 500 &mu;l FM, one tube for Axin2-high and the other for Axin2-low cells. Make sure collection tubes are in a chamber cooled to 4&deg;C during the sort.</li>
	<li>Record number of cells counted by flow cytometer for each population.</li>
</ol>
4. Representative Results (see Figure 2):
With the above protocol, we typically recover 40-45% debris-free cells from both the wildtype and Axin2LacZ/+ cochleae (Figures 2A and B), with about 70% of them free of doublets (see Figures 2A' and B'). Most sorted cells are viable and did not take up PI (~95%) (Figures 2A'' and B''). On average, the top ~20% CUG-positive cells are considered Axin2-LacZ-positive cells (Figure 2B''') and correlate to about 15,000 Axin2-LacZ-high cells per 10-12 animals.
Flow cytometry re-sort analysis of Axin2-high cells revealed greater than 93% purity in the Cascade Blue channel. In order to further confirm the identity of these cells, we performed post-sort immunostaining of both Axin2-high and Axin2-low cells using anti-&beta;-galactosidase antibodies, and found that the Axin2-high cells contained ~93.4% &beta;- galactosidase-positive cells (greater than 1,500 cells analyzed) and that Axin2-low cells contained ~4.8% &beta;-galactosidase-positive cells.
Figures and Tables:
<img alt="Figure 1" src="/files/ftp_upload/3432/3432fig1.jpg" /> 
Figure 1.Experimental paradigm. The above figure depicts the overall experimental strategy and techniques as described through the protocol section. Cochleae from wildtype and Axin2LacZ/+ mice are harvested and dissociated into single cells, which were then treated with CUG, a fluorescent substrate for &beta;-galactosidase. Axin2-LacZ-high and -low cells were then separated out via flow cytometry. <a href="https://www.jove.com/files/ftp_upload/3432/3432fig1large.jpg">Click here to view a full-sized version of this image.</a>
<img alt="Figure 2" src="/files/ftp_upload/3432/3432fig2.jpg" /> 
Figure 2.Gating Strategies. This figure illustrates the flow cytometry gating strategies used. A) Dissociated wildtype cells that underwent the same treatment as Axin2-LacZ cells are analyzed here using a total of 10,000 events. The first panel demonstrates the side scatter area (SSC-A) versus forward scatter area (FSC-A). Here, a distinct group of events are of a much larger size than those events closer to the y-axis. These events are designated as the "Debris Negative." The next gate, A', analyzes the "Debris Negative" cells from panel A, which is 43.8% of the total number of events (see table below). In this panel, the forward scatter height (FSC-H) versus the FSC-A is used to exclude doublets. Events deviating from the expected linear nature of the plot are excluded. This preparation typically yields ~70% events within the designated gate, and the remaining cells are excluded. In panel A'', propidium iodide (PI) as detected by the PE-Cy5 channel, is used to label dead cells (5.3%). In the next panel (A'''), the Cascade blue channel is used to detect the fluorescent substrate CUG. Here, the LacZ-high gates include less than 1% of total events. B) Dissociated cells from Axin2-LacZ mice treated with CUG and PI are analyzed here using a total of 10,000 events. Identical gates from panel A are applied here. B' shows that 29.5% of cells are excluded as doublets while B'' demonstrates that 5.4% are not viable. The initial gates for LacZ-high and -low cells are drawn using panel B''' with LacZ-high initially set at 19.7% and LacZ-low at 20.4%. <a href="https://www.jove.com/files/ftp_upload/3432/3432fig2large.jpg">Click here to view a full-sized version of this image.</a>
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Event number</td>
			<td>Percentage</td>
		</tr>
		<tr>
			<td>A</td>
			<td>Wildtype Analysis</td>
			<td>10,000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A'</td>
			<td>Debris Negative</td>
			<td>4,381</td>
			<td>43.80%</td>
		</tr>
		<tr>
			<td>A''</td>
			<td>Doublet Negative</td>
			<td>3,116</td>
			<td>71.10%</td>
		</tr>
		<tr>
			<td>A'''</td>
			<td>PI Negative</td>
			<td>2,951</td>
			<td>94.70%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>LacZ-high</td>
			<td>16</td>
			<td>0.54%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>LacZ-low</td>
			<td>2,790</td>
			<td>94.50%</td>
		</tr>
		<tr>
			<td>B</td>
			<td>Axin2-LacZ Analysis</td>
			<td>10,000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B'</td>
			<td>Debris Negative</td>
			<td>4,218</td>
			<td>42.2%</td>
		</tr>
		<tr>
			<td>B''</td>
			<td>Doublet Negative</td>
			<td>2,973</td>
			<td>70.50%</td>
		</tr>
		<tr>
			<td>B'''</td>
			<td>PI Negative</td>
			<td>2,813</td>
			<td>94.6%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>LacZ-high</td>
			<td>553</td>
			<td>19.70%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>LacZ-low</td>
			<td>573</td>
			<td>20.40%</td>
		</tr>
	</tbody>
</table>
Table 1. Quantitative analyses of gating strategies in Figure 2.

<ol>
	<li>Li, H., Liu, H., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+from+the+adult+mouse+inner+ear.">Pluripotent stem cells from the adult mouse inner ear.</a> Nat. Med.. 9, 1293-1299 (2003).</li><li>Oshima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+distribution+of+stem+cells+in+the+auditory+and+vestibular+organs+of+the+inner+ear.">Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.</a> J. Assoc. Res. Otolaryngol. 8, 18-31 (2007).</li><li>Barker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Jaks, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5+marks+cycling,+yet+long-lived,+hair+follicle+stem+cells.">Lgr5 marks cycling, yet long-lived, hair follicle stem cells.</a> Nat. Genet. 40, 1291-1299 (2008).</li><li>Kalani, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt-mediated+self-renewal+of+neural+stem/progenitor+cells.">Wnt-mediated self-renewal of neural stem/progenitor cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 16970-16975 (2008).</li><li>Willert, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+proteins+are+lipid-modified+and+can+act+as+stem+cell+growth+factors.">Wnt proteins are lipid-modified and can act as stem cell growth factors.</a> Nature. 423, 448-452 (2003).</li><li>Zeng, Y. A., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+proteins+are+self-renewal+factors+for+mammary+stem+cells+and+promote+their+long-term+expansion+in+culture.">Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture.</a> Cell Stem Cell. 6, 568-577 .</li><li>Chai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Expression+of+Lgr5,+a+Wnt+Target+Gene,+in+the+Developing+and+Mature+Mouse+Cochlea.">Dynamic Expression of Lgr5, a Wnt Target Gene, in the Developing and Mature Mouse Cochlea.</a> J. Assoc. Res. Otolaryngol. , .</li><li>Lustig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+feedback+loop+of+Wnt+signaling+through+upregulation+of+conductin/axin2+in+colorectal+and+liver+tumors.">Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors.</a> Mol. Cell Biol. 22, 1184-1193 (2002).</li><li>Diensthuber, M., Oshima, K., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem/progenitor+cells+derived+from+the+cochlear+sensory+epithelium+give+rise+to+spheres+with+distinct+morphologies+and+features.">Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features.</a> J. Assoc. Res. Otolaryngol. 10, 173-190 (2009).</li><li>Savary, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+stem/progenitor+cells+from+a+postnatal+cochlea+respond+to+Jagged1+and+demonstrate+that+notch+signaling+promotes+sphere+formation+and+sensory+potential.">Cochlear stem/progenitor cells from a postnatal cochlea respond to Jagged1 and demonstrate that notch signaling promotes sphere formation and sensory potential.</a> Mech. Dev. 125, 674-686 (2008).</li><li>White, P. M., Doetzlhofer, A., Lee, Y. S., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cochlear+supporting+cells+can+divide+and+trans-differentiate+into+hair+cells.">Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.</a> Nature. 441, 984-987 (2006).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+growth+and+differentiation+of+hair+cell+progenitors+from+the+newborn+rat+cochlear+greater+epithelial+ridge.">Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.</a> J. Neurosci. Methods. 164, 271-279 (2007).</li></ol>

No conflicts of interest declared.

Independent research studies have characterized limited regenerative capacity within the neonatal cochleae2,10,12,13. Using a GFP-reporter mice and flow cytometry, White and colleagues isolated specific cochlear supporting cells and found them to have progenitor cell characteristics12.
The canonical Wnt pathway has been demonstrated to mark stem/progenitor cell populations in multiple organ systems including the brain, mammary gland, hematopoietic system, skin, and gastro...

<table border="1">
<tr>
<td>Name of the reagent></td>
<td>Company</td>
<td>Catalog number</td>
<td>Comments </td>
</tr>
<tr>
<td>Petri dish, polystyrene, sterile Geriner Bio-one 35 x 10 mm </td>
<td>VWR</td>
<td>82050-540 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BD Falcon Cell Strainers, Sterile, BD Biosciences, blue, 40 &micro;m </td>
<td>VWR</td>
<td>21008-949 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Hanks&rsquo; Balanced Salt Solution (HBSS).&nbsp; Solution with Calcium, Magnesium, and without Phenol Red, Sterile </td>
<td>VWR</td>
<td>45000-456 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 50 ml </td>
<td>VWR </td>
<td>82050-346 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 15 ml </td>
<td>VWR</td>
<td>82050-278 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>B-27 Serum-Free Supplement (50x), liquid </td>
<td>Invitrogen</td>
<td>17504-044 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>N-2 Supplement (100x), liquid </td>
<td>Invitrogen</td>
<td>17502-048 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>bFGF, fibroblast growth factor-basic human </td>
<td>Sigma</td>
<td>F0291 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>IGF-1, insulin-like growth factor-1 from mouse </td>
<td>Sigma</td>
<td>I8779</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>EGF, epidermal growth factor human </td>
<td>Sigma</td>
<td>E9644 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dulbecco&rsquo;s Modified Eagle&rsquo;s Medium/Ham&rsquo;s F-12 50/50 Mix: 1X, with L-Glutamine&nbsp; and 15 mM HEPES</td>
<td>VWR</td>
<td>45000-350</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BD Falcon Round-Bottom Tubes, Disposable, Polystyrene, 12x75</td>
<td>VWR</td>
<td>60819-295</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trypsin, 0.25% (1X) with EDTA 4Na, liquid</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>ep Dualfilter TIPS 300uL, 960 TIPS</td>
<td>Eppendorf</td>
<td>22491245</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trypsin Inhibitor, Soybean, Purified</td>
<td>Worthington Biochem</td>
<td>LS003570</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BD Falcon 40um Cell Strainers</td>
<td>&nbsp;BD Biosciences</td>
<td>21008-949</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Multiwell Plates, Polystyrene, Greiner Bio-One, Nontreated Plates, 6 wells</td>
<td>VWR</td>
<td>82050-846</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Marker Gene FACS Blue LacZ beta-Galactosidase Detection Kit</td>
<td>Marker Gene Technologies</td>
<td>M0255</td>
<td>&nbsp;</td>
</tr>
</table>

isolating lacz-expressing cells from mouse inner ear tissues using flow cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Watch this Scientific Journal Video about Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry at JoVE.com

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Aislar las células que expresan LacZ de tejidos de ratón del oído interno mediante citometría de flujo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

isolating-lacz-expressing-cells-from-mouse-inner-ear-tissues-using

Research

JoVE Journal

Neuroscience

11.1K vistas.  Stanford University School of Medicine. Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of β-galactosidase prior to separation via flow cytometry.

Video: Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Aislar las células madre olfativo nasal de los roedores o los seres humanos

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Investigación

Neurociencias

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferencia de genes a la oreja del ratón en desarrollo por el interior<em&gt; En Vivo</em&gt; La electroporación

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Grabaciones de patch clamp en Inner células del oído pelo Aislado de pez cebra

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método quirúrgico para la entrega de genes mediada por virus de la oreja del ratón Interior a través de la ventana de membrana Ronda

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Análisis de las células inmunes en solo de los nervios ciático y de ganglio de raíz Dorsal de un ratón mediante citometría de flujo

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Quantification of Reactive Oxygen Species Using 2&amp;#8242;,7&amp;#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&amp;#252;ller Glial Cells

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

Cuantificación de especies reactivas de oxígeno utilizando sonda de dicacetato de 2′,7′-diclorofluoresceína y citometría de flujo en células gliales de Müller

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Purificación e identificación de proteínas específicas de tipo celular a partir de tejidos complejos utilizando una línea de ratón de ARNt sintetasa de metionina mutante

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

Cuantificación de la modificación de histonas en el cerebro del ratón mediante citometría de flujo

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Autor destacado: Mejoras en la investigación de la regulación de la expresión génica

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Cuantificación de las modificaciones postraduccionales globales de histonas mediante citometría de flujo intranuclear en microglía cerebral de ratón aislada

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...