Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei
在脊椎动物脑部采用荧光标记新鲜细胞核进行细胞周期分析
发布: 2013年11月20日第3卷第22期 DOI: 10.21769/BioProtoc.973 浏览次数: 17283
评审: Xuecai Ge
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
Abstract
Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.
Keywords: Flow cytometry (流式细胞仪)
DNA content (DNA含量)
Ploidy (倍性)
Tetraploid neuron (四倍体细胞)
Diploid neuron (二倍体细胞)
Materials and Reagents
- Frozen tissue samples (adult mouse hemicortex)
- Phosphate-buffered saline (PBS)
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Protease inhibitors cocktail tablets (cOmplete, Mini, EDTA-free) (Roche Diagnostics, catalog number: 11 836 170 001 )
- Bovine serum albumin (BSA) (stock 30 mg/ml in PBS) (Sigma-Aldrich, catalog number: A4503 )
- Calf serum (Life Technologies, catalog number: 16170-086 )
- Primary antibodies:
Mouse Anti-NeuN antibody (clone A60) (EMD Millipore, catalog number: mab377 )
Rabbit anti-EGR1 antibody (clone T.126.1) (Thermo Fisher Scientific, catalog number: MA5-15009 ) - Secondary antibodies:
Alexa Fluor 647 Goat Anti-Mouse IgG (H + L) Antibody (Life Technologies, catalog number: A21236 )
Alexa Fluor 488 Donkey Anti-Rabbit IgG (H + L) Antibody (Life Technologies, catalog number: A21206 ) - Propidium iodide (stock 1 mg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: P4170 )
- RNase A (stock 1 mg/ml, inactivated by boiling as indicated by the manufacturer) (Sigma-Aldrich, catalog number: R5000 )
- DAPI (stock 100 μg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: D9542 )
Equipment
- 15 ml tube
- 1.5 ml Eppendorf tube
- 7 ml Dounce homogenizer (WHEATON, model: 357542 )
- Microscope
- Refrigerated centrifuge
- Autoclaved 40 μm nylon filters
- Emission filters: BP 530/30, BP 616/23, BP 660/20, APC, FITC
- FACSAria cytometer (BD Biosciences) equipped with a double argon (488 nm) and helium-neon (633 nm) laser
Software
- FACSDiva (BD Biosciences)
- Modfit LT software (Verity Software)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Lopez-Sanchez, N. and Frade, J. M. (2013). Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei. Bio-protocol 3(22): e973. DOI: 10.21769/BioProtoc.973.
-
López-Sánchez, N. and Frade, J. M. (2013). Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33(17): 7488-7500.
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分类
神经科学 > 细胞机理
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
细胞生物学 > 细胞器分离 > 细胞核
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