Isolating LacZ-exprimierenden Zellen aus Maus Innenohrgeweben mittels Durchflusszytometrie
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.).
Materials
| Name of the reagent> | Company | Catalog number | Comments |
| Petri dish, polystyrene, sterile Geriner Bio-one 35 x 10 mm | VWR | 82050-540 | |
| BD Falcon Cell Strainers, Sterile, BD Biosciences, blue, 40 µm | VWR | 21008-949 | |
| Hanks’ Balanced Salt Solution (HBSS). Solution with Calcium, Magnesium, and without Phenol Red, Sterile | VWR | 45000-456 | |
| Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 50 ml | VWR | 82050-346 | |
| Cellstar centrifuge tubes, polypropylene, sterile, greiner bio-one, 15 ml | VWR | 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 | F0291 | |
| IGF-1, insulin-like growth factor-1 from mouse | Sigma | I8779 | |
| EGF, epidermal growth factor human | Sigma | E9644 | |
| Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 50/50 Mix: 1X, with L-Glutamine and 15 mM HEPES | VWR | 45000-350 | |
| BD Falcon Round-Bottom Tubes, Disposable, Polystyrene, 12×75 | VWR | 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 Biochem | LS003570 | |
| BD Falcon 40um Cell Strainers | BD Biosciences | 21008-949 | |
| Multiwell Plates, Polystyrene, Greiner Bio-One, Nontreated Plates, 6 wells | VWR | 82050-846 | |
| Marker Gene FACS Blue LacZ beta-Galactosidase Detection Kit | Marker Gene Technologies | M0255 |
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