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FACS purification of immunolabeled cell types from adult rat brain.

ABSTRACT: Molecular analysis of brain tissue is greatly complicated by having many different classes of neurons and glia interspersed throughout the brain. Fluorescence-activated cell sorting (FACS) has been used to purify selected cell types from brain tissue. However, its use has been limited to brain tissue from embryos or transgenic mice with promoter-driven reporter genes. To overcome these limitations, we developed a FACS procedure for dissociating intact cell bodies from adult wild-type rat brains and sorting them using commercially available antibodies against intracellular and extracellular proteins. As an example, we isolated neurons using a NeuN antibody and confirmed their identity using microarray and real time PCR of mRNA from the sorted cells. Our FACS procedure allows rapid, high-throughput, quantitative assays of molecular alterations in identified cell types with widespread applications in neuroscience.

SUBMITTER: Guez-Barber D

PROVIDER: S-EPMC3221768 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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FACS purification of immunolabeled cell types from adult rat brain.

Guez-Barber Danielle D Fanous Sanya S Harvey Brandon K BK Zhang Yongqing Y Lehrmann Elin E Becker Kevin G KG Picciotto Marina R MR Hope Bruce T BT

Journal of neuroscience methods 20110903 1

Molecular analysis of brain tissue is greatly complicated by having many different classes of neurons and glia interspersed throughout the brain. Fluorescence-activated cell sorting (FACS) has been used to purify selected cell types from brain tissue. However, its use has been limited to brain tissue from embryos or transgenic mice with promoter-driven reporter genes. To overcome these limitations, we developed a FACS procedure for dissociating intact cell bodies from adult wild-type rat brains ...[more]

PMID: 21911005

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Project description:RATIONALE:Quantifying cellular proteins in ventricular myocytes (MCs) is challenging due to tissue heterogeneity and the variety of cell sizes in the heart. In post-weaning cardiac ontogeny, rod-shaped MCs make up the majority of the cardiac mass while remaining a minority of cardiac cells in number. Current biochemical analyses of cardiac proteins do not correlate well the content of MC-specific proteins to cell type or size in normally developing tissue. OBJECTIVE:To develop a new large-particle fluorescent-activated cell sorting (LP-FACS) strategy for the purification of adult rod-shaped MCs. This approach is developed to enable growth-scaled measurements per-cell of the MC proteome and sarcomeric proteins (i.e. myosin heavy chain (MyHC) and alpha-actin (?-actin)) content. METHODS AND RESULTS:Individual cardiac cells were isolated from 21 to 94days old mice. An LP-FACS jet-in-air system with a 200-?m nozzle was defined for the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded ?95% purity of adult MCs independently of cell morphology and size. This approach excluded other cell types and tissue contaminants from further analysis. MC proteome, MyHC and ?-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient ?, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (?=1.02) and global protein accumulation (?=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and ?-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (?=1.79 and 2.19 respectively) and MC volumes (?=1.76 and 1.45 respectively). CONCLUSION:Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and ?-actin are concentrated more rapidly than what would be expected from MC proteome accumulation, cell enlargement, or animal growth alone. LP-FACS provides a new standard for adult MC purification and an approach to scale the biochemical content of specific proteins or group of proteins per cell in enlarging MCs.

Dataset Information

FACS purification of immunolabeled cell types from adult rat brain.

Publications

FACS purification of immunolabeled cell types from adult rat brain.

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