Assessment of Mitochondrial DNA Content and Mass in Macrophages
巨噬细胞中线粒体DNA含量和质量的评估
发布: 2016年05月05日第6卷第9期 DOI: 10.21769/BioProtoc.1796 浏览次数: 12122
评审: Ivan ZanoniShannon RuppertAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Jul 2015
Abstract
Mitochondria are essential regulators in not only ATP generation and metabolic reprogramming but also the generation of reactive oxygen species (ROS) in response to pathogenic stimuli. During exposure to environmental stresses including oxidative stress, exercise, cell division and caloric restriction, mitochondria can be divided to increase mitochondrial number, size, and mass. Moreover, mitochondrial biogenesis has a crucial role in the resolution of inflammation through preserving metabolic function. Recently, diverse biochemical methods have been utilized to evaluate activity of mitochondrial biogenesis. In this protocol, we will describe an in vitro assay to measure mitochondrial DNA content and mass. Quantitative real-time PCR analysis for determination of mitochondrial DNA content is a powerful tool with the addition of flow cytometry or confocal microscopy for evaluating mitochondrial mass. Together, these protocols may provide the significant information for mitochondria studies.
Keywords: Mitochondrial DNA content (线粒体DNA含量)
Macrophages (巨噬细胞)
Mitochondrial DNA Mass (线粒体DNA的质量)
Quantitative real-time PCR analysis (实时定量PCR分析)
Flow cytometry (流式细胞仪)
Materials and Reagents
- Microcentrifuge tube (1.5 ml or 2 ml)
- Lipopolysaccharide (InvivoGen, catalog number: tlrl-3pelps )
- G-DEXTM IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, catalog number: 17231 )
- Absolute or 70% ethanol (Merck Millipore Corporation, catalog number: 100983 )
- Isopropanol (Sigma-Aldrich, catalog number: I9030 )
- DEPC water (Merck Millipore Corporation, Calbiochem, catalog number: 9062-500 ML_CN )
- Primer (EC21, Solgent Co)
Stock concentration: 100 μM; Working concentration: 10 μM- mND-1 (mitochondrially encoded NADH dehydrogenase 1)
forward: 5’ GGCTACATACAATTACGCAAAG-3’
reverse: 5’-TAGAATGGAGTAGACCGAAAGG-3’ - mPyruvate kinase
forward: 5’ ACTGGCCGGTGTCATAGTGA-3’
reverse: 5’-TGTTGACCAGCCGTATGGATA-3’
- mND-1 (mitochondrially encoded NADH dehydrogenase 1)
- SYBR green PCR master mix (QIAGEN, catalog number: 330520 )
- MitoTracker Green FM (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M-7514 )
- Accutase (Sigma-Aldrich, catalog number: A6964 )
- Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 70011044 )
- Formaldehyde for cell fixation (Sigma-Aldrich, catalog number: F8755 )
- Triton® X-100 (Sigma-Aldrich, catalog number: T8787 )
- DMSO (Sigma-Aldrich, catalog number: D8418 )
- DMEM (Thermo Fisher Scientific, catalog number: 11965 )
- Macrophage Colony-Stimulating Factor (M-CSF) from mouse (Sigma-Aldrich, catalog number: M9170 )
- Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
- EDTA solution (Sigma-Aldrich, catalog number: 03690 )
- Heat-inactivated FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 1600044 )
- Penicillin (50 U/ml)/Streptomycin (50 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
- TE buffer (see Recipes)
- M-CSF-containing medium (see Recipes)
Equipment
- Tabletop microcentrifuge (KITA, Hanil, model: Micro12 )
- DNA electrophoresis chamber (Takara Bio Company, catalog number: AD140 )
- Qiagen Rotor gene 6000 instrument for quantitative real-time PCR analysis (Takara Bio Company, catalog number: AD140)
- BD FACSCantoTM II flow cytometer (BD Biosciences)
- 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, catalog number: 3111 )
- Centrifuge (LaboGene, catalog number: i730R )
- Water bath or heat block for heating at 65 °C
- Vortex mixer (PRONEER, model: MX-S )
Software
- FlowJo (Tree Star Inc)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yuk, J., Kim, T. S. and Jo, E. (2016). Assessment of Mitochondrial DNA Content and Mass in Macrophages. Bio-protocol 6(9): e1796. DOI: 10.21769/BioProtoc.1796.
Download Citation in RIS Format
分类
免疫学 > 免疫细胞功能 > 巨噬细胞
分子生物学 > DNA > DNA 提取
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
