בידוד LacZ-להביע תאים פנימית עכבר רקמות האוזן באמצעות זרימת cytometry

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 potential^1,2. 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 systems^3-7. 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 described^2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Protocol

1. Microdissection of the cochlea Prepare dissection microscope under hood by wiping surfaces with 70% ethanol and setting out ice blocks. 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). Prepare two 35 mm Petri dishes under sterile condition and fill half-way (~2 ml) with sterile 1x phosphate buffered solution (PBS). Sterilize micro-forceps in 50 ml conical Falcon tube with 70% etha…

Discussion

Independent research studies have characterized limited regenerative capacity within the neonatal cochleae^2,10,12,13. 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…

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