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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   

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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

  1. Microcentrifuge tube (1.5 ml or 2 ml)
  2. Lipopolysaccharide (InvivoGen, catalog number: tlrl-3pelps )
  3. G-DEXTM IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, catalog number: 17231 )
  4. Absolute or 70% ethanol (Merck Millipore Corporation, catalog number: 100983 )
  5. Isopropanol (Sigma-Aldrich, catalog number: I9030 )
  6. DEPC water (Merck Millipore Corporation, Calbiochem, catalog number: 9062-500 ML_CN )
  7. Primer (EC21, Solgent Co)
    Stock concentration: 100 μM; Working concentration: 10 μM
    1. mND-1 (mitochondrially encoded NADH dehydrogenase 1)
      forward: 5’ GGCTACATACAATTACGCAAAG-3’
      reverse: 5’-TAGAATGGAGTAGACCGAAAGG-3’
    2. mPyruvate  kinase
      forward: 5’ ACTGGCCGGTGTCATAGTGA-3’
      reverse: 5’-TGTTGACCAGCCGTATGGATA-3’
  8. SYBR green PCR master mix (QIAGEN, catalog number: 330520 )
  9. MitoTracker Green FM (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M-7514 )
  10. Accutase (Sigma-Aldrich, catalog number: A6964 )
  11. Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 70011044 )
  12. Formaldehyde for cell fixation (Sigma-Aldrich, catalog number: F8755 )
  13. Triton® X-100 (Sigma-Aldrich, catalog number: T8787 )
  14. DMSO (Sigma-Aldrich, catalog number: D8418 )
  15. DMEM (Thermo Fisher Scientific, catalog number: 11965 )
  16. Macrophage Colony-Stimulating Factor (M-CSF) from mouse (Sigma-Aldrich, catalog number: M9170 )
  17. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
  18. EDTA solution (Sigma-Aldrich, catalog number: 03690 )
  19. Heat-inactivated FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 1600044 )
  20. Penicillin (50 U/ml)/Streptomycin (50 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  21. TE buffer (see Recipes)
  22. M-CSF-containing medium (see Recipes)

Equipment

  1. Tabletop microcentrifuge (KITA, Hanil, model: Micro12 )
  2. DNA electrophoresis chamber (Takara Bio Company, catalog number: AD140 )
  3. Qiagen Rotor gene 6000 instrument for quantitative real-time PCR analysis (Takara Bio Company, catalog number: AD140)
  4. BD FACSCantoTM II flow cytometer (BD Biosciences)
  5. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, catalog number: 3111 )
  6. Centrifuge (LaboGene, catalog number: i730R )
  7. Water bath or heat block for heating at 65 °C
  8. Vortex mixer (PRONEER, model: MX-S )

Software

  1. FlowJo (Tree Star Inc)

Procedure

  1. Quantification of mitochondrial DNA content
    1. Prepare bone marrow-derived macrophages (BMDM) in 24 well plates (1 x 106 cells per each well, medium volume: 1 ml). Please refer to Chen (2012).
    2. Briefly, primary BMDM are differentiated for 5 days in M-CSF-containing medium.
    3. Remove the M-CSF-containing medium and then wash the adherent cells with 500 μl PBS three times.
    4. Add a fresh culture medium to exclude diverse alternative effects by M-CSF and FBS.
    5. After 24 h, BMDM were stimulated with lipopolysaccharide (LPS, 100 ng/ml) for the various time points in the incubator at 37 °C (Figure 1).
    6. Remove the supernatant and then wash the adherent cells in 500 μl PBS.
    7. Remove PBS leaving behind the adherent cells.
    8. Add 300 μl cell lysis buffers, pipette three times to lyse the adherent cells, and then transfer into a 1.5 ml microcentrifuge tube.
    9. Add 1.5 μl RNase A solution to the 1.5 ml tube containing the cell lysate and then incubate for 30 min in a heat block at 37 °C.
    10. Chill samples on ice for 5 min, add 100 μl protein precipitation (PPT) buffer into each sample, and vigorously mix by a vortex mixer for 30 sec.
    11. Centrifuge at 16,000 x g for 5 min and transfer 300 μl of the supernatants into the a new 1.5 ml tube.
    12. Add 300 μl of 100% isopropanol and then gently mix the samples containing the DNA and protein pellet by inverting several times.
    13. Centrifuge at 16,000 x g for 1 min, remove the supernatants, and then add 1 ml 70% ethanol to wash DNA (white pellet) in each tube.
    14. Centrifuge at 16,000 x g for 1 min, remove the ethanol, and dry the DNA pellet by leaving the cap open for 10 min.
    15. Incubate the DNA samples into 100 μl DNA rehydration buffer at 65 °C for 1 h using a water bath or heat block. In this case, we generally obtained 10-20 μg DNA from 1 x 106 cells.
    16. Prepare DNA mixture for Real-Time PCR analysis as follows:
      1. 10 μl 2x SYBR green PCR master mix
      2. 2 μl DNA samples (50 ng/μl)
      3. 1 μl Forward primer (10 μM)
      4. 1 μl Reverse primer (10 μM)
      5. 6 μl DEPC water
    17. Perform quantitative Real-Time PCR cycling follows:
      1. 95 °C for 10 min
      2. 40 cycles
        95 °C for 10 sec
        60 °C for 30 sec
        72 °C for 30 sec
        72 °C for 10 min
      3. 4 °C hold
    18. The quantification of mitochondrial DNA contents was calculated by the mitochondrial (mND-1) to nuclear DNA (mPyruvate kinase) ratio (Figure 1).


      Figure 1. TLR4 stimulation resulted in enhanced mitochondrial DNA content in macrophages. BMDMs were stimulated with LPS (100 ng/ml) for the indicated durations. Cells were collected and then analyzed by quantitative real-time PCR for measuring mitochondrial DNA content in BMDMs. The mitochondrial DNA content (mND-1) was normalized to the nuclear DNA (mPyruvate kinase).

  2. Mitochondrial DNA mass
    1. Preparation of bone marrow-derived macrophages (BMDM) in 24 well plate (1 x 106 cells per each well, medium volume: 1 ml).
    2. Remove the supernatant and then add 1 ml fresh culture medium in each well.
    3. After 24 h, BMDM cells are stimulated with LPS (100 ng/ml) for 6 h.
    4. Remove the supernatant and wash the adherent cells with 500 μl fresh PBS 3 times.
    5. Add 500 μl culture medium containing MitoTracker Green FM (Working concentration: 200 nM).
    6. Incubate the samples at 37 °C for 45~60 min.
    7. Remove the culture medium and wash the cells with 500 μl fresh PBS.
    8. Add 500 μl accutase into each sample.
    9. Transfer each sample to the FACS tube and centrifuge at 16,000 x g for 5 min.
    10. Remove the supernatants and wash the cells with 500 μl fresh PBS.
    11. Carefully replace the PBS with pre-warmed growth medium containing 3.7% formaldehyde at 37 °C for 15 min or at 4 °C overnight.
    12. Total fluorescence of MitoTracker Green FM is measured by flow cytometry (Figure 2).


      Figure 2. TLR4 stimulation resulted in enhanced mitochondrial DNA content in macrophages. BMDMs were stimulated with LPS (100 ng/ml) for 6 h. Cells were collected and then assessed by a flow cytometry analysis to measure Mitotracker fluorescence signals (Left). Live cells were assigned according to size and granularity (Right). Histogram of MitoTracker Green FM signal indicates the cell counts that correspond to the fluorescence intensity of mitochondrial mass.

Recipes

  1. TE buffer
    10 mM Tris-HCl
    0.1 mM EDTA
  2. M-CSF-containing medium
    DMEM
    10% heat-inactivated FBS
    Penicillin (50 U/ml)/Streptomycin (50 mg/ml)

Acknowledgments

The protocol was adapted from Experimental Procedures of previously published paper in Yuk et al. (2015). This work was supported by research fund of Chungnam National University.

References

  1. Chen, R. (2012). Isolation and culture of mouse bone marrow-derived macrophages (BMM’phi’). Bio-protocol 2(3): e68.
  2. Yang, C. S., Kim, J. J., Kim, T. S., Lee, P. Y., Kim, S. Y., Lee, H. M., Shin, D. M., Nguyen, L. T., Lee, M. S., Jin, H. S., Kim, K. K., Lee, C. H., Kim, M. H., Park, S. G., Kim, J. M., Choi, H. S. and Jo, E. K. (2015). Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome. Nat Commun 6: 6115.
  3. Yuk, J. M., Kim, T. S., Kim, S. Y., Lee, H. M., Han, J., Dufour, C. R., Kim, J. K., Jin, H. S., Yang, C. S., Park, K. S., Lee, C. H., Kim, J. M., Kweon, G. R., Choi, H. S., Vanacker, J. M., Moore, D. D., Giguere, V. and Jo, E. K. (2015). Orphan nuclear receptor ERRalpha controls macrophage metabolic signaling and A20 expression to negatively regulate TLR-Induced inflammation. Immunity 43(1): 80-91.

简介

线粒体是不仅ATP生成和代谢重新编程,而且响应病原性刺激的活性氧(ROS)的生成的必要调节器。在暴露环境应激,包括氧化应激,运动,细胞分裂和热量限制期间,线粒体可以分为增加线粒体数量,大小和质量。此外,线粒体生物发生在通过保护代谢功能解决炎症中具有关键作用。最近,不同的生物化学方法已被用来评估线粒体生物发生的活性。在这个协议,我们将描述一个体外测定来测量线粒体DNA含量和质量。用于测定线粒体DNA含量的定量实时PCR分析是添加流式细胞术或共聚焦显微镜用于评价线粒体质量的有力工具。在一起,这些协议可以提供线粒体研究的重要信息。

关键字:线粒体DNA含量, 巨噬细胞, 线粒体DNA的质量, 实时定量PCR分析, 流式细胞仪

材料和试剂

  1. 微量离心管(1.5ml或2ml)
  2. 脂多糖(InvivoGen,目录号:tlrl-3pelps)
  3. G-DEX TM IIc基因组DNA提取试剂盒(iNtRON Biotechnology,目录号:17231)
  4. 绝对或70%乙醇(Merck Millipore Corporation,目录号:100983)
  5. 异丙醇(Sigma-Aldrich,目录号:I9030)
  6. DEPC水(Merck Millipore公司,Calbiochem,目录号:9062-500ML_CN)
  7. 底漆(EC21,Solgent Co)
    库存浓度:100μM;工作浓度:10μM
    1. mND-1(线粒体编码的NADH脱氢酶1)
      正向:5'GGCTACATACAATTACGCAAAG-3'
      反向:5'-TAGAATGGAGTAGACCGAAAGG-3'
    2. mPyruvate 激酶
      正向:5'ACTGGCCGGTGTCATAGTGA-3'
      反向:5'-TGTTGACCAGCCGTATGGATA-3'
  8. SYBR green PCR主混合物(QIAGEN,目录号:330520)
  9. MitoTracker Green FM(Thermo Fisher Scientific,Molecular Probes TM,目录号:M-7514)
  10. Accutase(Sigma-Aldrich,目录号:A6964)
  11. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:70011044)
  12. 用于细胞固定的甲醛(Sigma-Aldrich,目录号:F8755)
  13. Triton X-100(Sigma-Aldrich,目录号:T8787)
  14. DMSO(Sigma-Aldrich,目录号:D8418)
  15. DMEM(Thermo Fisher Scientific,目录号:11965)
  16. 来自小鼠的巨噬细胞集落刺激因子(M-CSF)(Sigma-Aldrich,目录号:M9170)
  17. Tris-HCl(Sigma-Aldrich,目录号:T5941)
  18. EDTA溶液(Sigma-Aldrich,目录号:03690)
  19. 热灭活的FBS(Thermo Fisher Scientific,Gibco TM ,目录号:1600044)
  20. 青霉素(50U/ml)/链霉素(50mg/ml)(Thermo Fisher Scientific,Gibco公司,目录号:15140-122)
  21. TE缓冲区(参见配方)
  22. 含M-CSF的培养基(见配方)

设备

  1. 台式微量离心机(KITA,Hanil,型号:Micro12)
  2. DNA电泳室(Takara Bio Company,目录号:AD140)
  3. 用于定量实时PCR分析的Qiagen Rotor基因6000仪器(Takara Bio Company,目录号:AD140)
  4. BD FACSCanto TM II流式细胞仪(BD Biosciences)
  5. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,目录号:3111)。
  6. 离心机(LaboGene,目录号:i730R)
  7. 用于在65℃下加热的水浴或加热块
  8. 涡旋混合器(PRONEER,型号:MX-S)

软件

  1. FlowJo(Tree Star Inc)

程序

  1. 线粒体DNA含量的定量
    1. 在24孔板中制备骨髓来源的巨噬细胞(BMDM)(每孔1×10 6个细胞,培养基体积:1ml)。请参考陈(2012)。
    2. 简言之,在含有M-CSF的培养基中将原代BMDM分化5天
    3. 取出含M-CSF的培养基,然后用500μlPBS洗涤贴壁细胞三次。
    4. 添加新鲜培养基以排除M-CSF和FBS的多种替代效应
    5. 24小时后,在37℃下在培养箱中的不同时间点用脂多糖(LPS,100ng/ml)刺激BMDM(图1)。
    6. 除去上清液,然后在500μlPBS中洗涤贴壁细胞
    7. 取出PBS留下粘附细胞。
    8. 加入300微升细胞裂解缓冲液,移液器三次,以裂解贴壁细胞,然后转移到Microcentrifuge 1.5毫升管。
    9. 添加1.5微升RNA酶A溶液到含有细胞裂解液的1.5毫升管,然后在加热块在37℃孵育30分钟。
    10. 在冰上冷却样品5分钟,向每个样品中加入100μl蛋白质沉淀(PPT)缓冲液,并通过涡旋混合器剧烈混合30秒。
    11. 以16,000×g离心5分钟,并将300μl上清液转移到1.5ml新管中。
    12. 加入300微升的100%异丙醇,然后通过倒置几次轻轻混合含有DNA和蛋白质沉淀的样品
    13. 以16,000×g离心1分钟,除去上清液,然后加入1ml 70%乙醇以洗涤每个管中的DNA(白色沉淀)。
    14. 以16,000×g离心1分钟,除去乙醇,并通过将盖子打开10分钟来干燥DNA沉淀。
    15. 使用水浴或加热块,将DNA样品在65℃孵育1小时至100μlDNA再水化缓冲液中。在这种情况下,我们通常从1×10 6个细胞获得10-20μgDNA。
    16. 准备DNA混合物进行实时PCR分析如下:
      1. 10μl2x SYBR green PCR主混合物
      2. 2μlDNA样品(50 ng /μl)
      3. 1μl正向引物(10μM)
      4. 1μl反向引物(10μM)
      5. 6μlDEPC水
    17. 定量实时PCR的循环如下进行:
      1. 95℃10分钟
      2. 40个周期
        95°C 10秒
        60℃,30秒
        72℃30秒
        72℃10分钟
      3. 4°C保持
    18. 线粒体DNA含量的定量通过线粒体(mND-1)与核DNA(mPyruvate激酶)比率计算(图1)。


      图1. TLR4刺激导致巨噬细胞中线粒体DNA含量增加。在指定的持续时间用LPS(100ng/ml)刺激BMDM。收集细胞,然后通过定量实时PCR分析以测量BMDM中的线粒体DNA含量。线粒体DNA含量(mND-1)相对于核DNA(mPyruvate激酶)标准化。

  2. 线粒体DNA质量
    1. 在24孔板中制备骨髓来源的巨噬细胞(BMDM)(每孔1×10 6个细胞,培养基体积:1ml)。
    2. 除去上清液,然后在每个孔中加入1ml新鲜培养基。
    3. 24小时后,用LPS(100ng/ml)刺激BMDM细胞6小时
    4. 取出上清液,并在500μl新鲜的PBS中洗涤贴壁细胞3次
    5. 加入500μl含有MitoTracker Green FM(工作浓度:200nM)的培养基
    6. 将样品在37℃孵育45?60分钟
    7. 取出培养基,并在500μl新鲜的PBS中洗涤细胞
    8. 向每个样品中加入500μlaccutase
    9. 转移到FACS管并在16,000×g离心5分钟
    10. 取出上清液,并在500μl新鲜PBS中洗涤细胞
    11. 小心地取出PBS,并将其与含有3.7%甲醛的预热生长培养基在37℃下更换15分钟或在4℃过夜。
    12. 通过流式细胞术测量MitoTracker Green FM的总荧光(图2)。


      图2. TLR4刺激导致巨噬细胞中线粒体DNA含量增加。用LPS(100ng/ml)刺激BMDM 6小时。收集细胞,然后通过流式细胞术分析来测量Mitotracker荧光信号(左)。根据大小和粒度分配活细胞(右)。 MitoTracker直方图绿色FM信号表示对应于线粒体质量的荧光强度的细胞计数。

食谱

  1. TE缓冲区
    10mM Tris-HCl
    0.1mM EDTA
  2. 含M-CSF的培养基 DMEM
    10%热灭活的FBS 青霉素(50U/ml)/链霉素(50mg/ml)

致谢

该方案改编自Yuk等人先前发表的论文的实验程序。 (2015)。这项工作是由庆南国立大学的研究基金支持。

参考文献

  1. Chen,R。(2012)。 小鼠骨髓来源的巨噬细胞(BMM'phi')的分离和培养生物-protocol 2(3):e68。
  2. Yang,CS,Kim,JJ,Kim,TS,Lee,PY,Kim,SY,Lee,HM,Shin,DM,Nguyen,LT,Lee,MS,Jin,HS,Kim,KK, MH,Park,SG,Kim,JM,Choi,HSand Jo,EK(2015)。 小型异源二聚体配偶体与NLRP3相互作用,并负调节NLRP3炎症小体的活化。 Nat Commun 6:6115.
  3. Yuk,JM,Kim,TS,Kim,SY,Lee,HM,Han,J.,Dufour,CR,Kim,JK,Jin,HS,Yang,CS,Park,KS,Lee,CH,Kim,JM,Kweon ,GR,Choi,HS,Vanacker,JM,Moore,DD,Giguere,V。和Jo,EK(2015)。 孤儿核受体ERRalpha控制巨噬细胞代谢信号传导,A20表达负调节TLR诱导的炎症。 a> 免疫力 43(1):80-91
  • English
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
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