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 JoVE Neuroscience

フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

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Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine

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Cite this Article: フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Jan, T. A., Chai, R., Sayyid, Z. N., Cheng, A. G. Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry. J. Vis. Exp. (58), e3432, doi:10.3791/3432 (2011).

Disclosures: フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Materials: フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Name Company Catalog Number Comments
Petri dish, polystyrene, sterile Geriner Bio-one 35 x 10 mm

VWR international 82050-540

BD Falcon Cell Strainers, Sterile, BD Biosciences, blue, 40 m

VWR international 21008-949

Hanks’ Balanced Salt Solution (HBSS). Solution with Calcium, Magnesium, and without Phenol Red, Sterile

VWR international 45000-456

Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 50 ml

VWR international 82050-346

Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 15 ml

VWR international 82050-278

B-27 Serum-Free Supplement (50x), liquid

Invitrogen 17504-044

N-2 Supplement (100x), liquid

Invitrogen 17502-048

bFGF, fibroblast growth factor-basic human

Sigma-Aldrich F0291

IGF-1, insulin-like growth factor-1 from mouse

Sigma-Aldrich I8779
EGF, epidermal growth factor human

Sigma-Aldrich E9644

Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 50/50 Mix: 1X, with L-Glutamine and 15 mM HEPES

 VWR international 45000-350
BD Falcon Round-Bottom Tubes, Disposable, Polystyrene, 12x75

 VWR international 60819-295
Trypsin, 0.25% (1X) with EDTA 4Na, liquid

 Invitrogen 25200-056
ep Dualfilter TIPS 300uL, 960 TIPS

 Eppendorf 22491245
Trypsin Inhibitor, Soybean, Purified

 Worthington Biochemical LS003570
BD Falcon 40um Cell Strainers

 BD Biosciences 21008-949
Multiwell Plates, Polystyrene, Greiner Bio-One, Nontreated Plates, 6 wells

 VWR international 82050-846
Marker Gene FACS Blue LacZ beta-Galactosidase Detection Kit

 Marker Gene Technologies M0255

References: フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

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

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