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A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

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Related in: MedlinePlus

Dissociation of vestibular sensory epithelia into single hair cells and non-sensory cells. AM1-43-stained sensory epithelia underwent different enzymatic and non-enzymatic treatments to test for optimal conditions to separate hair cells and non-sensory cells and to preserve hair cell morphology. Conditions were: 0.25% trypsin (A), 0.05% trypsin (B), accutase (C), accumax (D), enzyme-free (E) and 50% accumax + 0.025% trypsin (F). Shown are representative images of cells after mild trituration following 7 min incubations at 37°C.
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pone-0066026-g002: Dissociation of vestibular sensory epithelia into single hair cells and non-sensory cells. AM1-43-stained sensory epithelia underwent different enzymatic and non-enzymatic treatments to test for optimal conditions to separate hair cells and non-sensory cells and to preserve hair cell morphology. Conditions were: 0.25% trypsin (A), 0.05% trypsin (B), accutase (C), accumax (D), enzyme-free (E) and 50% accumax + 0.025% trypsin (F). Shown are representative images of cells after mild trituration following 7 min incubations at 37°C.

Mentions: We optimized the dissociation method for vestibular sensory epithelia to ensure thorough cell separation and minimal cell aggregation but also high viability judged by cell shape and exclusion of propidium iodide, a dye that is generally unable to enter viable cells. Representative results obtained with different dissociation strategies are shown in Figure 2. The predominantly enzymatic digestion conditions included 0.25% trypsin, Accutase cell detachment mixture, an enzyme-free formulation of chelating reagents, 0.05% trypsin, Accumax cell dissociation mixture, and 50% Accumax plus 0.025% trypsin. We found that neither trypsin alone nor the commercially available enzyme cocktails Accutase or Accumax were satisfactory to quantitatively dissociate the tissue. These tests were systematically done by varying incubation times from a few minutes to up-to 30 minutes, followed by mild trituration, and resulted in either insufficient cell separation or starkly reduced viability (Fig. 2A–D). Combining Accumax cell dissociation solution at half strength with a low concentration of trypsin, however, for a total incubation time of 7 minutes resulted in the optimal separation of viable individual cells (Fig. 2F). Hair cells separated with this procedure displayed at least some rudimentary preservation of cytomorphology. The enzyme-free formulation of chelating reagents alone was also highly efficient for cell separation (Fig. 2E); however, hair cell morphology was not well preserved in this condition, presumably caused by chelating divalent cations such as Ca2+, which are important for hair bundle integrity [16], [17]. Moreover, cell viability was reduced when compared with the Accumax and trypsin combination.


A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Dissociation of vestibular sensory epithelia into single hair cells and non-sensory cells. AM1-43-stained sensory epithelia underwent different enzymatic and non-enzymatic treatments to test for optimal conditions to separate hair cells and non-sensory cells and to preserve hair cell morphology. Conditions were: 0.25% trypsin (A), 0.05% trypsin (B), accutase (C), accumax (D), enzyme-free (E) and 50% accumax + 0.025% trypsin (F). Shown are representative images of cells after mild trituration following 7 min incubations at 37°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g002: Dissociation of vestibular sensory epithelia into single hair cells and non-sensory cells. AM1-43-stained sensory epithelia underwent different enzymatic and non-enzymatic treatments to test for optimal conditions to separate hair cells and non-sensory cells and to preserve hair cell morphology. Conditions were: 0.25% trypsin (A), 0.05% trypsin (B), accutase (C), accumax (D), enzyme-free (E) and 50% accumax + 0.025% trypsin (F). Shown are representative images of cells after mild trituration following 7 min incubations at 37°C.
Mentions: We optimized the dissociation method for vestibular sensory epithelia to ensure thorough cell separation and minimal cell aggregation but also high viability judged by cell shape and exclusion of propidium iodide, a dye that is generally unable to enter viable cells. Representative results obtained with different dissociation strategies are shown in Figure 2. The predominantly enzymatic digestion conditions included 0.25% trypsin, Accutase cell detachment mixture, an enzyme-free formulation of chelating reagents, 0.05% trypsin, Accumax cell dissociation mixture, and 50% Accumax plus 0.025% trypsin. We found that neither trypsin alone nor the commercially available enzyme cocktails Accutase or Accumax were satisfactory to quantitatively dissociate the tissue. These tests were systematically done by varying incubation times from a few minutes to up-to 30 minutes, followed by mild trituration, and resulted in either insufficient cell separation or starkly reduced viability (Fig. 2A–D). Combining Accumax cell dissociation solution at half strength with a low concentration of trypsin, however, for a total incubation time of 7 minutes resulted in the optimal separation of viable individual cells (Fig. 2F). Hair cells separated with this procedure displayed at least some rudimentary preservation of cytomorphology. The enzyme-free formulation of chelating reagents alone was also highly efficient for cell separation (Fig. 2E); however, hair cell morphology was not well preserved in this condition, presumably caused by chelating divalent cations such as Ca2+, which are important for hair bundle integrity [16], [17]. Moreover, cell viability was reduced when compared with the Accumax and trypsin combination.

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH
Related in: MedlinePlus