2 users have reported that they have successfully carried out the experiment using this protocol.
Measurement of Mitochondrial DNA Release in Response to ER Stress

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Sep 2015



Mitochondria house the metabolic machinery for cellular ATP production. The mitochondrial network is sensitive to perturbations (e.g., oxidative stress and pathogen invasion) that can alter membrane potential, thereby compromising function. Healthy mitochondria maintain high membrane potential due to oxidative phosphorylation (Ly et al., 2003). Changes in mitochondrial function or calcium levels can cause depolarization, or a sharp decrease in mitochondrial membrane potential (Bernardi, 2013). Mitochondrial depolarization induces opening of the mitochondrial permeability transition pore (MPTP), which allows release of mitochondrial components like reactive oxygen species (mtROS), mitochondrial DNA (mtDNA) or intermembrane space proteins into the cytosol (Martinou and Green, 2001; Tait and Green, 2010; Bronner and O'Riordan, 2014). These contents trigger inflammation, and can lead to cell death (West et al., 2011). Both mtROS and cytosolic mtDNA contribute to the activation of inflammasomes, multiprotein complexes that process the proinflammatory cytokines, IL-18 and IL-1β. Studies indicate that cytosolic mtDNA in particular can bind two different inflammasome sensors, AIM2 and NLRP3, leading to inflammasome activation (Burckstummer et al., 2009; Hornung and Latz, 2010). In this protocol, you will be able to specifically extract cytosolic mtDNA and quantify the amount using a qPCR assay.

Figure 1. Flowchart for extracting, purifying, and amplifying cytosolic mtDNA

Part I. Extraction and purification of cytosolic mtDNA

Materials and Reagents

  1. For extraction (see Figure 1)
    1. 1.5 ml microcentrifuge tubes (Denville Scientific Inc., catalog number: C2170 )
    2. Gloves
    3. 6 well-plates, tissue culture-treated (Corning, catalog number: 3506 )
    4. Cell lifter (Biologix Group Limited, catalog number: 70-2180 )
    5. Murine immortalized bone marrow derived macrophages (Bernardi, 2013)
    6. Thapsigargin, 97%, ACROS OrganicsTM (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: AC328570010 ) (Bronner et al., 2015)
    7. 1% NP-40 (Igepal CA-630) (Sigma-Aldrich, catalog number: I8896 ) (Bronner and O'Riordan, 2014)
    8. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-133 ) (Burckstummer, 2009)
    9. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
    10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
    11. Medium (see Recipes)
    12. NP-40 (Igepal CA-630) solution (see Recipes)
    13. Thapsigargin stock solution (see Recipes)

  2. For purification
    1. Gloves
    2. DNeasy blood & tissue kit, 50 samples (QIAGEN, catalog number: 69504 ) or 250 samples (QIAGEN, catalog number: 69506 )
    3. Ethanol 200 proof (Decon Labs, catalog number: 2716 )


  1. Centrifuge (Thermo Fisher Scientific, Fisher ScientificTM, model: accuSpinTM Micro 17 )


  1. Seed 1 x 106 cells in 2 ml per well into 6-well plate in the appropriate medium.
  2. The following day (~16 h later), aspirate medium and add medium containing thapsigargin (10 µM in 2 ml medium for 4-6 h) (Hornung and Latz, 2010).
  3. At relevant times post-treatment (e.g., 2 h, 4 h and 8 h) wash cells with 1x DPBS once then aspirate 1x DPBS.
  4. Add 1% NP-40 (100 µl) to each well and scrape cells.
  5. Place lysates into prelabeled microcentrifuge tubes and incubate on ice for 15 min (Livak and Schmittgen, 2001).
  6. Spin lysates at 13,000 rpm (16,000 x g) for 15 min at 4 °C to pellet the insoluble fraction.
  7. Transfer supernatant (the cytosolic fraction) to a new tube and discard the pellet.
    1. Use supernatant in the next step to extract cytosolic mitochondrial DNA.
  8. Use DNeasy Blood & Tissue Kit to purify mitochondrial DNA from the cytosolic fraction according to the manufacturer’s instructions (Ly et al., 2003).
    1. Add 100 µl ethanol (96-100%) to the cytosolic fraction and continue to step 4 in the DNeasy Blood & Tissue Kit protocol.


  1. This protocol is optimized for using immortalized bone marrow derived macrophages (iBMDM) (Bronner et al., 2015). Protocol can be optimized for chosen experimental cell type.
  2. Thapsigargin serves as a positive control for triggering mitochondrial DNA release. Thapsigargin prevents the uptake of calcium into the endoplasmic reticulum (ER) by blocking SERCA channels. Under these conditions, the ER leaks calcium without being able to replenish its calcium stores, and consequently calcium accumulates in the cytosol or mitochondria. Mitochondrial calcium overload triggers depolarization, leading to the release mitochondrial contents into the cytosol.
  3. Make a 10% NP-40 stock solution and dilute to 1% NP-40.
  4. Use DPBS that contains calcium and magnesium to ensure that cells will remain attached during the wash step.
  5. Prelabeled microcentrifuge tubes do not need to be prechilled.
  6. 1 x 106 to 1 x 107 (seeded in 100 mm tissue culture dishes) cells will yield 1-20 µg of mtDNA.


  1. Medium
    10% Fetal Bovine Serum (FBS)
    Add 50 ml heat-inactivated FBS to 450 ml of DMEM.
    1. FBS is heat inactivated for 30 min at 55 °C.
    2. This medium has been optimized for iBMDM. Use medium optimized for experimental cell type.
  2. NP-40 (Igepal CA-630) solution
    1. For 10% NP-40, add 1 ml of NP-40 to 9 ml of dH2O. 
    2. For 1% NP-40, add 1 ml of 10% NP-40 to 9 ml of dH2O.
  3. Thapsigargin stock solution
    1. The concentration stated above has been optimized for iBMDM. The concentration used on different cell types must be optimized.
    2. Stock solution of thapsigargin remains usable up to one year after reconstitution and can be aliquoted and stored at -20 °C
    3. Stock solution of thapsigargin is 5 mM (1 mg in 307 µl DMSO). Dilute thapsigargin (4 µl into 2 ml of medium) into medium that will be added to the wells.

Part II. Amplification of mtDNA via qPCR
After extracting DNA from the cytosolic fraction, quantitative PCR is employed to measure cytosolic mitochondrial DNA.

Materials and Reagents

  1. 96 well qPCR plate (Denville Scientific Inc., catalog number: C18096) (Bernardi, 2013)
  2. 1.5 ml microcentrifuge tubes (Denville Scientific Inc., catalog number: C2170 )
  3. Gloves
  4. Brilliant II SYBR® green with low ROX (Agilent Technologies, catalog number: 600830 )
  5. Sterilized double distilled water
  6. Primers (see sequences below)


  1. qPCR machine (Stratagene MX3000P)


  1. Prepare the SYBR qPCR master reaction mix as follows in Table 1 for mitochondrial genes of interest and internal control (housekeeping gene for used here is 18S rDNA) in 1.5 ml microcentrifuge tubes:

    Table 1. Master Mix recipe for quantifying mtDNA release into cytosol via qPCR

    The following primers were used:
    Cytochrome c oxidase I (mt gene) 
    18S rDNA (internal control)
  2. Mix gently but thoroughly by pipetting.
  3. Incubate reaction mix on ice until ready to use.
  4. Aliquot DNA samples (1 µl) into wells (run in triplicates) (Bronner et al., 2015).
  5. Aliquot corresponding Master Mix (19 µl) into the corresponding wells (Bronner and O'Riordan, 2014).
  6. Use the thermal cycler program as seen below in Table 2:

    Table 2. Thermal cycler program for mtDNA release qPCR

  7. Once qPCR is complete, calculate relative fold change in cytochrome c oxidase I from the Ct values.

Representative data

Adapted method for calculating relative fold change (Livak and Schmittgen, 2001) between thapsigargin-treated and untreated Ct values that are represented in Table 3:

Table 3. Representative values from mtDNA release qPCR

    1. ΔCt Treatment = Target gene - Reference gene
      ΔCt Treatment (thapsigargin) = 19.15 - 20.54
      ΔCt Control = Target gene - Reference gene
      ΔCt Control (untreated) = 26.16 - 26.18
    2. ΔΔCt = ΔCt Treatment - ΔCt Control
      ΔΔCt = -1.39 - (-0.02)
      ΔΔCt = -1.37
    3. Relative Fold Change = 2-(ΔΔCt)
      Relative Fold Change = 2-(-1.37)
      Relative Fold Change = 2.6 

This result indicates that thapsigargin treatment results in a 2.6 fold increase in cytosolic mtDNA compared to untreated (Figure 2).

Figure 2. Graph of representative data from Table 3. Graph depicts cytosolic mtDNA relative fold change seen in Thapsigargin treated iBMDM when compared to untreated iBMDM.


  1. Use plates that are optimized for qPCR machine.
  2. The total reaction volume is 20 µl. When adding DNA ensure the amount is consistent between samples.
    1. 10 ng and 2 µg represent the minimum and maximum amount of DNA to use for qRT-PCR analysis
  3. Pipette as accurately as possible during steps 4 and 5, since small variations in volumes can increase variability in the qPCR results.


We acknowledge financial support from the University of Michigan Rackham Graduate School (D. N. B.), the UM Genetics Training Program (D. N. B., GM007544). This research was supported by funding from the NIH to M. X. D. O. (AI101777). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
We acknowledge that this protocol was adapted (Nakahira et al., 2011) and modified for use with immortalized bone marrow macrophages infected by a bacterial pathogen.


  1. Bernardi, P. (2013). The mitochondrial permeability transition pore: a mystery solved? Front Physiol 4: 95.
  2. Bronner, D. N., Abuaita, B. H., Chen, X., Fitzgerald, K. A., Nunez, G., He, Y., Yin, X. M. and O'Riordan, M. X. (2015). Endoplasmic reticulum stress activates the inflammasome via NLRP3- and Caspase-2-driven mitochondrial damage. Immunity 43(3): 451-462.
  3. Bronner, D. N. and O'Riordan, M. X. (2014). A near death experience: Shigella manipulates host death machinery to silence innate immunity. EMBO J 33(19): 2137-2139.
  4. Burckstummer, T., Baumann, C., Bluml, S., Dixit, E., Durnberger, G., Jahn, H., Planyavsky, M., Bilban, M., Colinge, J., Bennett, K. L. and Superti-Furga, G. (2009). An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol 10(3): 266-272.
  5. Hornung, V. and Latz, E. (2010). Intracellular DNA recognition. Nat Rev Immunol 10(2): 123-130.
  6. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408.
  7. Ly, J. D., Grubb, D. R. and Lawen, A. (2003). The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8(2): 115-128.
  8. Martinou, J. C. and Green, D. R. (2001). Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2(1): 63-67.
  9. Nakahira, K., Haspel, J. A., Rathinam, V. A., Lee, S. J., Dolinay, T., Lam, H. C., Englert, J. A., Rabinovitch, M., Cernadas, M., Kim, H. P., Fitzgerald, K. A., Ryter, S. W. and Choi, A. M. (2011). Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 12(3): 222-230.
  10. Tait, S. W. and Green, D. R. (2010). Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11(9): 621-632.
  11. West, A. P., Shadel, G. S. and Ghosh, S. (2011). Mitochondria in innate immune responses. Nat Rev Immunol 11(6): 389-402.


线粒体代表细胞ATP生产的代谢机制。线粒体网络对可以改变膜电位,从而损害功能的扰动(氧化应激和病原体侵入)敏感。健康的线粒体由于氧化磷酸化维持高的膜电位(Ly等人,2003)。线粒体功能或钙水平的变化可导致去极化,或线粒体膜电位的急剧下降(Bernardi,2013)。线粒体去极化诱导线粒体通透性转换孔(MPTP)的开放,其允许线粒体组分如活性氧(mtROS),线粒体DNA(mtDNA)或膜间隙蛋白释放到细胞质中(Martinou和Green,2001; Tait和Green ,2010; Bronner和O'Riordan,2014)。这些内容触发炎症,并且可导致细胞死亡(West等人,2011)。 mtROS和细胞溶质mtDNA有助于炎症细胞的活化,多蛋白复合物,其处理促炎细胞因子,IL-18和IL-1β。研究表明,胞质mtDNA尤其可以结合两种不同的炎症小体传感器AIM2和NLRP3,导致炎症小体激活(Burckstummer等人,2009; Hornung和Latz,2010)。在此协议中,您将能够特异性提取细胞质mtDNA并使用qPCR测定法定量。




  1. 对于提取(参见图1)
    1. 1.5ml微量离心管(Denville Scientific Inc.,目录号:C2170)
    2. 手套
    3. 6孔板,组织培养处理(Corning,目录号:3506)
    4. 细胞提升器(Biologix Group Limited,目录号:70-2180)
    5. 小鼠永生化骨髓来源的巨噬细胞(Bernardi,2013)
    6. 毒胡萝卜素,97%,ACROS Organics TM(Thermo Fisher Scientific,Fisher Scientific ,目录号:AC328570010)(Bronner等人,2015年, )
    7. 1%NP-40(Igepal CA-630)(Sigma-Aldrich,目录号:I8896)(Bronner和O'Riordan,2014)
    8. DPBS(Thermo Fisher Scientific,Gibco TM ,目录号:14040-133)(Burckstummer,2009)
    9. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
    10. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10437-028)
    11. 中等(见配方)
    12. NP-40(Igepal CA-630)溶液(参见配方)
    13. 毒胡萝卜素储备溶液(参见配方)

  2. 用于纯化
    1. 手套
    2. DNeasy血& 组织试剂盒,50个样品(QIAGEN,目录号:69504)或250个样品(QIAGEN,目录号:69506)
    3. 乙醇200proof(Decon Labs,目录号:2716)


  1. 离心机(Thermo Fisher Scientific,Fisher Scientific ,型号:accuSpinTM Micro 17)


  1. 以2ml /孔将1×10 6个细胞种植在6孔板中的适当培养基中。
  2. 第二天(约16小时后),吸出培养基并加入含有毒胡萝卜素的培养基(10μM在2ml培养基中培养4-6小时)(Hornung和Latz,2010)。
  3. 在治疗后(例如,2小时,4小时和8小时)的相关时间用1x DPBS洗涤细胞一次,然后吸出1×DPBS。
  4. 向每个孔中加入1%NP-40(100μl)并刮擦细胞
  5. 将裂解物置于预标记的微量离心管中,在冰上孵育15分钟(Livak和Schmittgen,2001)。
  6. 在4℃下以13,000rpm(16,000xg)旋转15分钟以使不溶性部分沉淀。
  7. 转移上清(细胞溶质部分)到新管,并丢弃沉淀。
    1. 使用上清液在下一步提取细胞质线粒体DNA。
  8. 使用DNeasy Blood& Tissue Kit,以根据制造商的说明书从细胞溶质级分纯化线粒体DNA(Ly et al。,2003)。
    1. 加入100μl乙醇(96-100%)到细胞溶质级分,并继续步骤4在DNeasy Blood& Tissue Kit协议。


  1. 该方案针对使用永生化骨髓来源的巨噬细胞(iBMDM)进行优化(Bronner等人,2015)。 协议可以针对所选择的实验细胞类型进行优化
  2. 毒胡萝卜素作为触发线粒体DNA释放的阳性对照。 毒胡萝卜素通过阻断SERCA通道阻止钙摄入内质网(ER)。 在这些条件下,ER渗漏钙,而不能补充其钙储备,因此钙积聚在细胞溶质或线粒体中。 线粒体钙超负荷触发去极化,导致线粒体内容物释放到胞质溶胶中
  3. 制成10%NP-40储备溶液并稀释至1%NP-40
  4. 使用含有钙和镁的DPBS,以确保细胞在洗涤步骤期间保持附着
  5. 预标记的微量离心管不需要预先冷却
  6. 1×10 6至1×10 7 sup/7(接种在100mm组织培养皿中)细胞将产生1-20μgmtDNA。


  1. 中等
    加入50毫升热灭活的FBS到450毫升DMEM 注意:
    1. FBS在55℃下热灭活30分钟。
    2. 此介质已针对iBMDM进行了优化。 使用针对实验细胞类型优化的培养基。
  2. NP-40(Igepal CA-630)溶液
    1. 对于10%NP-40,将1ml NP-40加入9ml dH 2 O中。
    2. 对于1%NP-40,将1ml 10%NP-40加入到9ml dH 2 O中。
  3. 毒胡萝卜素储备溶液
    1. 上述浓度已针对iBMDM进行了优化。 必须优化不同类型细胞的浓度。
    2. 毒胡萝卜素储备溶液在重构后可持续使用一年,可分装并储存在-20℃下
    3. 毒胡萝卜素的储备溶液是5mM(1mg在307μlDMSO中)。 稀释毒胡萝卜素(4微升,2毫升培养基),将添加到培养基中的孔。

第二部分。 放大 的mtDNA通过qPCR


  1. 96孔qPCR板(Denville Scientific Inc.,目录号:C18096)(Bernardi,2013)
  2. 1.5ml微量离心管(Denville Scientific Inc.,目录号:C2170)
  3. 手套
  4. Brilliant II SYBR ®绿色和低ROX(安捷伦科技公司,目录号:600830)
  5. 灭菌双蒸水
  6. 引物(见下面的序列)


  1. qPCR机(Stratagene MX3000P)


  1. 准备SYBR qPCR主反应混合物,如表1中所示的线粒体基因和内部控制(这里使用的管家基因是18S rDNA)在1.5ml微量离心管中:


    18S rDNA(内部对照)
  2. 轻轻混匀,用移液器充分混匀。
  3. 在冰上孵育反应混合物,直到准备使用。
  4. 将DNA样品(1μl)分装到孔中(一式三份)(Bronner等人,2015)。
  5. 将相应的Master Mix(19μl)分装到相应的孔中(Bronner和O'Riordan,2014)。
  6. 使用热循环仪程序,如下表2所示:

    表2. mtDNA释放qPCR的热循环程序

  7. 一旦qPCR完成,从Ct值计算细胞色素c氧化酶I的相对倍数变化。




    1. ΔCt治疗=目标基因 - 参考基因
      ΔCt处理(毒胡萝卜素)= 19.15-20.54
      ΔCtControl =目标基因 - 参考基因
      ΔCt对照(未处理)= 26.16-26.18
    2. ΔΔCt=ΔCt处理--ΔCt控制
      ΔΔCt= -1.39 - (-0.02)
      ΔΔCt= -1.37
    3. 相对折叠变化= 2 - (ΔΔCt)
      相对折叠变化= 2 - ( - 1.37)
      相对折叠变化= 2.6 




  1. 使用为qPCR机器优化的板
  2. 总反应体积为20μl。当添加DNA时,确保样品之间的量一致。
    1. 10 ng和2μg代表用于qRT-PCR分析的DNA的最小和最大量
  3. 在步骤4和5期间尽可能精确地移液,因为体积的小变化可以增加qPCR结果的可变性。




  1. Bernardi,P.(2013)。  线粒体通透性转变毛孔:一个神秘的解决了吗? 前生理 4:95
  2. Bronner,DN,Abuaita,BH,Chen,X.,Fitzgerald,KA,Nunez,G.,He,Y.,Yin,XM and O'Riordan,MX(2015)。  内质网应激通过NLRP3和Caspase-2驱动的线粒体损伤激活炎症小体。 免疫 43(3):451-462。
  3. Bronner,DN和O'Riordan,MX(2014)。  近死亡经验:Shigella操纵宿主死亡机制以沉默先天免疫。 EMBO J 33(19):2137-2139。
  4. Burckstummer,T.,Baumann,C.,Bluml,S.,Dixit,E.,Durnberger,G.,Jahn,H.,Planyavsky,M.,Bilban,M.,Colinge,J.,Bennett,KL和Superti -Furga,G。(2009)。 正交蛋白质组基因组筛选将AIM2鉴定为细胞质DNA传感器炎症小体。 Nat Immunol 10(3):266-272。
  5. Hornung,V。和Latz,E。(2010)。  Intracellular DNA recognition。 Nat Rev Immunol 10(2):123-130。
  6. Livak,KJ和Schmittgen,TD(2001)。  分析的相对基因表达数据,使用实时定量PCR和2(-DΔDelta C(T))方法。方法 25(4):402-408。
  7. Ly,JD,Grubb,DR和Lawen,A。(2003)。  凋亡中的线粒体膜电位(deltapsi(m));更新。细胞凋亡 8(2):115-128。
  8. Martinou,JC and Green,DR(2001)。  打破线粒体屏障。 Nat Rev Mol Cell Biol 2(1):63-67。
  9. Nabahira,K.,Haspel,JA,Rathinam,VA,Lee,SJ,Dolinay,T.,Lam,HC,Englert,JA,Rabinovitch,M.,Cernadas,M.,Kim,HP,Fitzgerald,KA,Ryter, SW和Choi,AM(2011)。  自噬蛋白调节通过抑制由NALP3炎症小体介导的线粒体DNA的释放而产生先天性免疫应答。自身免疫 12(3):222-230。
  10. Tait,SW and Green,DR(2010)。  Mitochondria和细胞死亡:外膜透化和超越。 Nat Rev Mol Cell Biol 11(9):621-632。
  11. West,AP,Shadel,GS和Ghosh,S。(2011)。  线粒体在先天免疫反应中。 Nat Rev Immunol 11(6):389-402。
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引用:Bronner, D. N. and O’Riordan, M. X. (2016). Measurement of Mitochondrial DNA Release in Response to ER Stress. Bio-protocol 6(12): e1839. DOI: 10.21769/BioProtoc.1839.