Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine
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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).
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.
1. Microdissection of the cochlea
2. Cell dissociation (modified from previously described methods)2,10
3. Flow Cytometry
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-β-galactosidase antibodies, and found that the Axin2-high cells contained ~93.4% β- galactosidase-positive cells (greater than 1,500 cells analyzed) and that Axin2-low cells contained ~4.8% β-galactosidase-positive cells.
Figures and Tables:
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 β-galactosidase. Axin2-LacZ-high and -low cells were then separated out via flow cytometry. Click here to view a full-sized version of this image.
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%. Click here to view a full-sized version of this image.
Table 1. Quantitative analyses of gating strategies in Figure 2.
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 gastrointestinal intestinal crypts. Recent work has found active Wnt signaling in a poorly characterized cell population in the neonatal cochlea8. The Axin2-LacZ reporter mouse9 generates a faithful readout of active Wnt signaling in the cochlea8 like in other systems5,7. In order to better characterize this population of Axin2-LacZ-positive cells, we modified and combined several established approaches to investigate the role of Wnt signaling in regulating regeneration in the neonatal cochlea.
The use of fluorescence-conjugated substrates to label LacZ-positive cochlear cells prior to cell sorting presents several technical challenges that would not be encountered when sorting fluorescent reporter (e.g., GFP-positive) cells from transgenic mice. We used a known fluorescent substrate of β-galactosidase called 3-carboxyumbelliferyl β-D-galactopyranoside (CUG)5,7. However, this substrate is not specific to the E. coli β-galactosidase enzyme and endogenous murine β-galactosidase can also be labeled in a dose-dependent manner. To maximize substrate specificity, we determined the level of nonspecific labeling by processing wildtype cochleae with the same CUG staining procedure in each experiment. This control experiment is necessary to create a threshold for setting gates used to identify Axin2-high cells, which we defined as the most fluorogenic cells from the Axin2-LacZ cochleae, especially since this protocol typically generates a "shoulder" of LacZ-positive cells (see Figure 1B''').
Another challenging aspect of this technique is the paucity of cells in the cochlea, making isolation of rare subpopulations difficult. In order to overcome this, we simply increased the number of mice utilized for each experiment (20 to 50 mice). One of the critical steps is therefore to maintain cell viability prior to undergoing the stress of cell sorting. The following three steps were found important in optimizing cell viability: 1) Minimize dissection time: we coordinated a team of dissectors (2-3 people) to shorten the duration of harvesting cells. We routinely completed dissections of 80 cochleae in less than two hours with a team of 3 investigators; 2) Gentle trituration: it is important to perform this step on non-coated 6-well plates using a blunt tip pipette in a gentle manner; 3) Keep dissociated cells on ice: we found that keeping tissues/cells on ice in as many steps as possible improved subsequent cell viability. Gating of viable cells during sorting must remain consistent among different experiments as the final line is selected by the operator. We determined our set value (seen in Figure 2A'') following analysis of more than 500,000 events (data not shown) on one plot where the viable and non-viable cells demonstrate two separate foci on dot plot.
During the sorting process, an experienced flow cytometer user is needed to maintain standards and voltages constant in different trials. More importantly, it is essential to become familiar with the cochlear cells using one flow cytometer (one machine) to avoid re-configurations of voltages during every experiment and thus potential variability. This will not only save time, but will also allow for faster processing of cells and thereby increase cell viability. To further increase viability, we found the use of a 100 μm nozzle superior to the 70 μm nozzle. Following isolation of Axin2-high and Axin2-low cells, one can proceed with a number of different experiments, including gene array analyses or cell culture assays.
In conclusion, we have established a novel and reliable technique to separate out LacZ-expressing cells from the neonatal cochlea using flow cytometry. This protocol aims to facilitate the isolation and characterization of rare stem/progenitor cell populations. Future efforts include improving cell sorting efficiency to decrease the number of experimental animals needed.
No conflicts of interest declared.
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.).
|Petri dish, polystyrene, sterile Geriner Bio-one 35 x 10 mm||VWR international||82050-540|
|BD Falcon Cell Strainers, Sterile, BD Biosciences, blue, 40 Ám||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|