Name: isolating lacz-expressing cells from mouse inner ear tissues using flow cytometry
Uploaded: 2011-12-23T13:00:00
Description: Watch this Scientific Journal Video about Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry at JoVE.com

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% etha...

<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>

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.

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 ...

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