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Oxidative Stress Assays (arsenite and tBHP) in Caenorhabditis elegans
秀丽隐杆线虫中氧化应激指标(亚砷酸盐和tBHP)的测定   

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Abstract

Cells and organisms face constant exposure to reactive oxygen species (ROS), either from the environment or as a by-product from internal metabolic processes. To prevent cellular damage from ROS, cells have evolved detoxification mechanisms. The activation of these detoxification mechanisms and their downstream responses represent an overlapping defense response that can be tailored to different sources of ROS to adequately adapt and protect cells. In this protocol, we describe how to measure the sensitivity to oxidative stress from two different sources, arsenite and tBHP, using the nematode C. elegans.

Keywords: Hydrogen peroxide(过氧化氢), ROS(ROS), Xenobiotics(外源性物质), SKN-1(SKN-1), DAF-16(DAF-16)

Background

Reactive oxygen species (ROS) are small molecules that can damage DNA, proteins, lipids and other cellular components. Systemic levels of ROS induce irreversible cellular damage, which has been implicated in the etiology of aging and age-related diseases, such as Alzheimer’s disease, atherosclerosis, and diabetes. Furthermore, environmental toxins such as pollutants, smoke, chemicals, radiation, and xenobiotics significantly induce ROS formation. To protect against oxidative damage, cells have evolved complex mechanisms that detoxify ROS. Interestingly, long-lived animals show an enhancement of these protective mechanisms, implicating their importance for healthy aging.

The multicellular organism C. elegans has been instrumental in elucidating the molecular mechanisms that protect against ROS (Blackwell et al., 2015). In C. elegans, the major ROS detoxification mechanisms are initiated by the transcription factor SKN-1, the orthologue of the Nrf (nuclear factor-erythroid-related factor) proteins (Blackwell et al., 2015). Exposing C. elegans to either the metalloid sodium arsenite (As) or tert-Butyl hydroperoxide (tBHP; an organic peroxide) activates SKN-1, which promotes survival. Although overlapping sets of genes are upregulated by SKN-1 in response to As or tBHP, there are also condition-specific gene sets that tailor the oxidative stress response (Oliveira et al., 2009). Moreover, the expression of almost all detoxification genes in response to As depends on SKN-1, whereas the induction of several genes upon tBHP-treatment is also independent of SKN-1 (Oliveira et al., 2009), suggesting the activation of other oxidative stress response transcription factors. How different ROS sources are sensed and integrated is not well understood, but recently a mechanism has been elucidated for how As-induced ROS are generated and sensed by the cell (Hourihan et al., 2016).

Treating C. elegans with As activates the BLI-3/NADPH oxidase complex to produce localized pools of ROS, which modify a cysteine in the IRE-1 kinase and induce the SKN-1-dependent antioxidant response leading to lifespan extension (Hourihan et al., 2016). Further supporting the function of localized ROS levels as cell signals, recent work has identified a novel regulator of aging, MEMO-1, which increases resistance to As toxicity and facilitates lifespan extension in a BLI-3- and SKN-1-dependent manner (Ewald et al., 2017).

Taken together, oxidative stress responses can be induced directly, by exogenously added ROS sources such as tBHP, or as a secondary response to a chemical (such as As) or other stress leading to increased ROS levels. These two ROS sources elicit common, but also distinct downstream stress-response genes and protection mechanisms. Here, we describe the protocols for both ROS sources to assess C. elegans survival under these oxidative stress conditions.

Part I. Protocol for As stress tolerance assay

Materials and Reagents

  1. Pipette tips
  2. 24-well plates (Multiwell Culture Plate 24 well fl. NUNC, clear)
  3. C. elegans strains (available at Caenorhabditis Genetics Center [CGC] https://cbs.umn.edu/cgc/home)
  4. Nematode growth medium (NGM) culturing C. elegans plates (He, 2011) seeded with bacteria (OP50) grown overnight (300 μl OP50 per plate)
  5. M9 buffer (Physiological buffer for C. elegans; [He, 2011])
  6. Sodium arsenite solution volumetric, 0.05 M NaAsO2 (Honeywell International, catalog number: 35000 )
  7. Agar (He, 2011)
  8. Phosphate buffer (He, 2011)
  9. Calcium chloride (CaCl2) (He, 2011)
  10. Magnesium sulfate (MgSO4) (He, 2011)
  11. Cholesterol (He, 2011)

Equipment

  1. Pipettes
  2. Stereomicroscope
  3. Worm pick

Procedure

  1. Day 0 (Monday): Using the worm pick (Figure 1), transfer larval 4 stage (L4) worms onto fresh NGM culturing plate (He, 2011). Store these worms overnight at 20 °C.
    Note: You need about 50 L4 worms per strain; e.g., 10-12 for each of the triplicates with As and for the control condition (M9 buffer only).


    Figure 1. Material for the As-survival assay

  2. Day 1 (Tuesday): Put 50 μl of M9 buffer into each well of a 24-well plate. See Figure 2 for loading scheme. Place 10-12 one-day-old worms into the M9 (Figure 3 and Video 1). You need 3 wells per strain for As and 1 well for M9 buffer as a control. When all worms are set, fill wells with either 450 μl of 5.56 mM As (for a final concentration of 5 mM As dissolved in M9) or M9.
  3. Score every hour for worm survival. (Exploded animals need to be excluded from the statistics.)

    Important: The 5 mM As in M9 dilution must be prepared fresh directly before you put it into the wells.



    Figure 2. Loading scheme for the As response assay. WT = wild type, Mut = mutant.


    Figure 3. Transferring worms into the drop of M9 buffer in the 24-well plates
    Note: When transferring worms into the 50 μl M9 buffer drop in the well, worms may become injured by scratching the worms off the worm pick. Check and exclude non-moving worms (Video 1) before filling up wells with the As solution.

    Video 1. Loading C. elegans into 24-well plates for the arsenite oxidative stress assay. 10-12 C. elegans were transferred into a well of the 24-well plate that contains a 50 μl drop of M9. Worms should be freely trashing. Exclude non-moving worms (marked in Figure 3) before filling up wells with the As solution.

Data analysis

For As-assay, the estimates of the survival functions are calculated by using the product-limit (Kaplan-Meier) method (Figure 4 and Table 1). The log-rank (Mantel-Cox) method is used to test the null hypothesis and calculate P-values. Data were analyzed using JMP statistical software from SAS.


Figure 4. Survival plot of As-assay. Loss-of-function mutation in skn-1 (green curve) makes these animals more sensitive to 5 mM As, whereas reduction-of-function mutation in daf-2 (red curve) makes animals more resistant to 5 mM As (Ewald et al., 2015). For statistical details, please see Table 1.

Table 1. Statistics for As-assay


Part II. Protocol for tBHP oxidative stress assay

Materials and Reagents

  1. Pipette tips
  2. 6 cm Petri dishes (He, 2011)
  3. Protective gloves
  4. Luperox® TBH70X, tert-Butyl hydroperoxide solution 70% in H2O (Sigma-Aldrich, catalog number: 458139 )
    Note: tert-Butyl hydroperoxide (tBHP) is an organic peroxide widely used in a variety of oxidation processes. tBHP has an advantage over hydrogen peroxide in that it is less labile. However, tBHP plates should be stored in an airtight container and used within 24 h. tBHP is normally supplied as a 69-70% aqueous solution.
    Safety Warning: tert-Butyl hydroperoxide is an exceptionally dangerous chemical that is highly reactive, flammable and toxic. It is corrosive to skin and mucous membranes and causes respiratory distress when inhaled.
  5. Nematode growth medium (NGM) culturing C. elegans plates (He, 2011) seeded with bacteria (OP50) grown overnight (300 μl OP50 per plate)
  6. Agar (He, 2011)
  7. Phosphate buffer (He, 2011)
  8. Calcium chloride (CaCl2) (He, 2011)
  9. Magnesium sulfate (MgSO4) (He, 2011)
  10. Cholesterol (He, 2011)

Equipment

  1. Pipettes
  2. Face mask
  3. Safety goggles
  4. Worm pick
  5. Fume hood
  6. Stereomicroscope

Software

  1. SAS JMP statistical software

Procedure

  1. Day 0 (Monday): Pick L4 worms onto fresh NGM culturing plate (He, 2011) seeded with OP50 bacteria.
    Note: You need 60 L4 worms per strain; 20 worms per tBHP plate, triplicates.
  2. Day 1 (Tuesday): Prepare tBHP plates (see Recipe 1).
  3. Place agar in dH2O to get a 4% agar solution (100 ml agar = 6 plates) and heat up to solve. Let agar solution cool down (50-60 °C) until you can hold it with your hand and hold it against your wrist, put gloves on, then add all solutions (phosphate buffer, CaCl2, MgSO4, and cholesterol). Shake this briefly to avoid the formation of CaSO4 precipitate and in the end the tBHP in the fume hood. Pour the plates by hand (equal amounts, so that bottom is filled). Place the plates in an airtight box to seal them, and they will be ready for use the next day.
  4. Day 2 (Wednesday): Pick 20 two-day-old adults onto the tBHP plates (Figure 5). Remember to wear protective gloves, safety goggles, and face mask.


    Figure 5. Materials for the tBHP-survival assay

  5. Important: Worms will try to run off the plate (Figure 6). Hence, for the first two hours you need to shuffle worms back into the center of the plate. Therefore, immediately after you put the worms on the tBHP plates, start looking at your first plates again for ‘escaping worms’. We typically assay 4 strains in parallel allowing enough time to shuffle worms back into the center of all 12 plates in a reasonable time.
  6. Score survival every hour. (Exclude exploded animals from the statistics.)


    Figure 6. Scoring tBHP plates. A. Shows starting place of worms with a scoop of OP50 bacterial food that attracts them so that they stay a little bit longer in the center of the plate. B. Shows how worms start to run off the plates after several minutes. They try to get away from the tBHP in the plate and usually run off the agar onto the plastic of the petri dish, where they dry out. After two hours, wild-type worms on tBHP will cease crawling around.

Data analysis

For tBHP assay, the estimates of survival functions are calculated by using the product-limit (Kaplan-Meier) method (Figure 7 and Table 2). The log-rank (Mantel-Cox) method is used to test the null hypothesis and calculate P-values. Data were analyzed using JMP statistical software from SAS.
 

Figure 7. Survival plot of tBHP-assay. RNAi knockdown of daf-2 (red curve) makes wild-type animals more resistant to 15.4 mM tBHP compared to wild-type animals treated with L4440 empty vector control RNAi (Ewald et al., 2015).

Table 2. Statistics for tBHP-assay

Notes

We want to note that while the protocols described here work reproducibly well, many variations have previously been described, including whether the animals have been provided with food. Different variations can be found in papers from (An and Blackwell, 2003; Tullet et al., 2008; Oliveira et al., 2009; Wang et al., 2010; Robida-Stubbs et al., 2012). The protocols described here are based upon and optimized from this previous work, and have recently been used in (Ewald et al., 2015; Steinbaugh et al., 2015; Hourihan et al., 2016; Ewald et al., 2017).

Recipes

  1. tBHP plates
    100 ml 4% agar dissolved in dH2O
    2.5 ml phosphate buffer
    100 μl CaCl2
    100 μl MgSO4
    160 μl cholesterol
    214 μl tBHP [Stock = 70%] hence, final concentration = 15.4 mM

Acknowledgments

We thank the Blackwell and Ewald lab for developing and refining these assays. Picture and movie credit for Nadine Herrmann and Eline Jongsma (ETH Zurich).

References

  1. An, J. H. and Blackwell, T. K. (2003). SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev 17(15): 1882-1893.
  2. Blackwell, T. K., Steinbaugh, M. J., Hourihan, J. M., Ewald, C. Y. and Isik, M. (2015). SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med 88(Pt B): 290-301.
  3. Ewald, C. Y., Hourihan, J. M., Bland, M. S., Obieglo, C., Katic, I., Moronetti Mazzeo, L. E., Alcedo, J., Blackwell, T. K. and Hynes, N. E. (2017). NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans. Elife 6.
  4. Ewald, C. Y., Landis, J. N., Porter Abate, J., Murphy, C. T. and Blackwell, T. K. (2015). Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity. Nature 519(7541): 97-101.
  5. He, F. L. (2011). Common worm media and buffers. Bio-protocol Bio101: e55.
  6. Hourihan, J. M., Moronetti Mazzeo, L. E., Fernandez-Cardenas, L. P. and Blackwell, T. K. (2016). Cysteine sulfenylation directs IRE-1 to activate the SKN-1/Nrf2 antioxidant response. Mol Cell 63(4): 553-566.
  7. Oliveira, R. P., Porter Abate, J., Dilks, K., Landis, J., Ashraf, J., Murphy, C. T. and Blackwell, T. K. (2009). Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8(5): 524-541.
  8. Robida-Stubbs, S., Glover-Cutter, K., Lamming, D. W., Mizunuma, M., Narasimhan, S. D., Neumann-Haefelin, E., Sabatini, D. M. and Blackwell, T. K. (2012). TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. Cell Metab 15(5): 713-724
  9. Steinbaugh, M. J., Narasimhan, S. D., Robida-Stubbs, S., Moronetti Mazzeo, L. E., Dreyfuss, J. M., Hourihan, J. M., Raghavan, P., Operana, T. N., Esmaillie, R. and Blackwell, T. K. (2015). Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence. Elife 4.
  10. Tullet, J. M., Hertweck, M., An, J. H., Baker, J., Hwang, J. Y., Liu, S., Oliveira, R. P., Baumeister, R. and Blackwell, T. K. (2008). Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 132(6): 1025-1038.
  11. Wang, J., Robida-Stubbs, S., Tullet, J. M., Rual, J. F., Vidal, M. and Blackwell, T. K. (2010). RNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance and longevity deriving from translation inhibition. PLoS Genet 6(8).

简介

细胞和生物体从环境或内部代谢过程的副产物中经常暴露于活性氧(ROS)。 为了防止ROS细胞损伤,细胞已经发展出解毒机制。 这些解毒机制及其下游反应的激活代表了重叠的防御反应,可以针对不同的ROS来源进行调整,以充分适应和保护细胞。 在本协议中,我们描述了如何使用线虫C测量两种不同来源亚砷酸盐和tBHP对氧化应激的敏感性。线虫。
【背景】活性氧(ROS)是可以损害DNA,蛋白质,脂质和其他细胞成分的小分子。系统水平的ROS诱导不可逆的细胞损伤,其涉及衰老和年龄相关疾病如阿尔茨海默氏病,动脉粥样硬化和糖尿病的病因。此外,环境毒素如污染物,烟雾,化学物质,辐射和异生物质会显着诱导ROS形成。为了防止氧化损伤,细胞已经发展出复杂的ROS解毒机制。有趣的是,长寿的动物显示出这些保护机制的增强,暗示它们对健康老化的重要性。
 多细胞生物体C。线虫有助于阐明防止ROS的分子机制(Blackwell等人,2015)。在C.线虫,主要的ROS解毒机制由转录因子SKN-1(Nrf(核因子 - 红细胞相关因子)蛋白质的直向同源物)(Blackwell等人,2015)开始)。公开C。线虫与准金属亚砷酸钠(As)或叔丁基过氧化氢(tBHP;有机过氧化物)激活促进存活的SKN-1。虽然重叠的基因组被SKN-1响应于As或tBHP上调,但也存在着条件特异性基因组,其定制氧化应激反应(Oliveira等,2009)。此外,几乎所有解毒基因在As上的表达取决于SKN-1,而在tBHP治疗时几种基因的诱导也与SKN-1无关(Oliveira等人,2009) ),表明其他氧化应激反应转录因子的激活。感觉和整合的不同ROS来源不尽如人意,但是近来已经阐明了如何由细胞产生和感测As诱导的ROS的机制(Hourihan等人,2016)。
治疗 C。线虫与As激活BLI-3 / NADPH氧化酶复合物以产生局部的ROS池,其修饰IRE-1激酶中的半胱氨酸并诱导SKN-1依赖性抗氧化反应,导致寿命延长(Hourihan et al。,2016)。进一步支持局部ROS水平作为细胞信号的功能,最近的工作已经确定了一种新的老化调节因子MEMO-1,其增加了对As毒性的抗性并促进了BLI-3-和SKN-1依赖性方式的寿命延长( Ewald等人,2017)。
 总之,氧化应激反应可以通过外源性添加的ROS来源(如tBHP)或作为对化学物质(如As)的二次反应或导致ROS水平升高的其他应激来直接诱导。这两种ROS来源引起共同的,也是不同的下游应激反应基因和保护机制。在这里,我们描述两个ROS源的协议来评估C。这些氧化应激条件下的线虫存活。

关键字:过氧化氢, ROS, 外源性物质, SKN-1, DAF-16

第一部分作为耐应力测定的方案

材料和试剂

  1. 移液器提示
  2. 24孔板(Multiwell Culture Plate 24 well fl。NUNC,clear)
  3. ℃。线虫菌株(可在Caenorhabditis遗传学中心获得[CGC] https:/ /cbs.umn.edu/cgc/home
  4. 生长过夜的细菌(OP50)种植丝虫线虫生长培养基(NGM)培养线虫(He,2011)(每片300μlOP50)
  5. M9缓冲液(线虫的生理缓冲液; [他,2011])
  6. 亚砷酸钠溶液体积,0.05M NaAsO 2(Honeywell International,目录号:35000)
  7. 琼脂(他,2011)
  8. 磷酸盐缓冲液(He,2011)
  9. 氯化钙(CaCl 2)(He,2011)
  10. 硫酸镁(MgSO 4)(He,2011)
  11. 胆固醇(他,2011)

设备

  1. 移液器
  2. 立体显微镜
  3. 蠕虫选择

程序

  1. 第0天(星期一):使用蠕虫(图1),将幼虫4期(L4)虫传播到新鲜的NGM培养板上(He,2011)。将这些蠕虫在20°C保存过夜。
    注意:每个菌株需要约50只L4虫;例如,对于具有As和控制条件(仅限于M9缓冲器)的一式三份中的每一个,为10-12。


    图1. As-survival测定的材料

  2. 第1天(星期二):将50μl的M9缓冲液放入24孔板的每个孔中。加载方案见图2。将10-12只一天的蠕虫放入M9(图3和视频1)。对于As,每个菌株需要3个孔,对于M9缓冲液需要1个孔。当所有的蠕虫被设置时,用450微升的5.56mM的As(最终浓度为5mM,溶解在M9中)或M9填充孔。
  3. 得分每小时蠕虫存活。 (爆炸动物需要排除在统计数据之外。)

    重要提示:M9稀释液中的5 mM As必须在将其放入孔中之前直接重新制备。



    图2. As响应测定的载体方案。WT =野生型,Mut =突变体。


    图3.将蠕虫传送到24孔板中的M9缓冲液滴
    注意:当将蠕虫传送到井中的50μlM9缓冲液滴中时,蠕虫可能会从蠕虫搔抓上被损坏。在使用As解决方案填充井之前,请检查并排除不可移动的蠕虫(视频1)。

    Video 1. Loading C. elegans into 24-well plates for the arsenite oxidative stress assay. 10-12 C. elegans were transferred into a well of the 24-well plate that contains a 50 μl drop of M9. Worms should be freely trashing. Exclude non-moving worms (marked in Figure 3) before filling up wells with the As solution.

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数据分析

对于As测定,通过使用产品限制(Kaplan-Meier)方法(图4和表1)计算生存函数的估计。对数秩(Mantel-Cox)方法用于测试零假设并计算P 值。使用SAS的JMP统计软件分析数据。


图4. As-assay的生存情况。 skn-1中的功能丧失突变(绿色曲线)使得这些动物对5mM As更敏感,而daf-2中的功能下降突变< (红曲线)使得动物更能抵抗5mM As(Ewald等人,2015)。有关统计资料,请参阅表1.

表1.统计学分析


第二部分。 tBHP氧化应激测定方案

材料和试剂

  1. 移液器提示
  2. 6厘米培养皿(他,2011年)
  3. 防护手套
  4. Luperox TBH70X,叔丁基过氧化氢溶液70%H 2 O(Sigma-Aldrich,目录号:458139)
    注意:叔丁基过氧化氢(tBHP)是广泛用于各种氧化工艺的有机过氧化物。 tBHP具有超过过氧化氢的优点,因为它较不稳定。然而,tBHP板应储存在密封的容器中,并在24小时内使用。 tBHP通常作为69-70%的水溶液提供。
    安全警告:叔丁基过氧化氢是一种特别危险的化学物质,具有高反应性,易燃性和有毒性。它对皮肤和粘膜有腐蚀性,并在吸入时引起呼吸窘迫。
  5. 线虫生长培养基(NGM)培养。线虫(He,2011)接种细菌(OP50)生长过夜(每片300μlOP50)
  6. 琼脂(他,2011)
  7. 磷酸盐缓冲液(He,2011)
  8. 氯化钙(CaCl 2)(He,2011)
  9. 硫酸镁(MgSO 4)(He,2011)
  10. 胆固醇(他,2011)

设备

  1. 移液器
  2. 面罩
  3. 安全护目镜
  4. 蠕虫选择
  5. 通风柜
  6. 立体显微镜

软件

  1. SAS JMP统计软件

程序

  1. 第0天(星期一):选择L4蠕虫进入新鲜的NGM培养板(He,2011),种植有OP50细菌。
    注意:每个菌株需要60只L4虫;每个tBHP板20个蠕虫,一式三份。
  2. 第1天(星期二):准备tBHP板(参见方法1)。
  3. 将琼脂放在dH 2 O中以得到4%琼脂溶液(100ml琼脂= 6片)并加热至解决。让琼脂溶液冷却(50-60°C),直到用手握住手掌并握住手腕,戴上手套,然后加入所有溶液(磷酸盐缓冲液,CaCl 2,硫酸镁,硫酸镁<3>和胆固醇)。暂时摇动以避免CaSO 4沉淀物的形成,最后在通风橱中结束tBHP。用手倒入盘子(等量的,以便底部填充)。将板放在密封的盒子中以密封它们,并在第二天就可以使用。
  4. 第二天(星期三):选择20名两岁大人到tBHP板上(图5)。请记得戴防护手套,安全护目镜和面罩。


    图5. tBHP生存测定的材料

  5. 重要提示:蠕虫将尝试从板上排出(图6)。因此,在前两个小时,您需要将蠕虫洗回板材的中心。因此,在将蠕虫放在tBHP板上之后,请先重新查看您的第一块板,以“逃脱虫”。我们通常平行测定4株,足够的时间在合理的时间内将蠕虫洗回所有12个平板的中心。
  6. 每小时得分生存(从统计数据中排除爆炸动物。)


    图6.计分tBHP平板。 A.显示吸吮它们的OP50细菌食物勺的蠕虫的起始位置,以使它们在板的中心保持一点时间。 B.显示蠕虫几分钟后开始跑出板块。他们试图离开板中的tBHP,并通常将琼脂倒在培养皿的塑料上,在那里它们变干。两个小时后,tBHP上的野生型蠕虫将停止爬行。

数据分析

对于tBHP测定,通过使用产品限制(Kaplan-Meier)方法(图7和表2)计算生存函数的估计。对数秩(Mantel-Cox)方法用于测试零假设并计算P 值。使用SAS的JMP统计软件分析数据。
&nbsp;

图7. tBHP检测的生存情况 daf-2的RNAi敲低(红曲线)使得野生型动物比野生型更能抵抗15.4mM tBHP用L4440空载体控制RNAi处理的动物(Ewald等人,2015)。

表2. tBHP测定的统计学

笔记

我们想要注意的是,虽然这里描述的方案可重复性好,但是以前已经描述了许多变化,包括动物是否已经被提供食物。来自(An和Blackwell,2003; Tullet等人,2008; Oliveira等人,2009; Wang等人的论文中可以找到不同的变化。 ,2010; Robida-Stubbs等人,2012)。这里描述的协议基于并优化了以前的工作,并且最近已被用于(Ewald等人,2015; Steinbaugh等人,2015年; Hourihan 等,,2016; Ewald等人,2017)。

食谱

  1. tBHP板
    将100ml 4%琼脂溶解在dH 2 O中 2.5毫升磷酸盐缓冲液
    100μlCaCl 2
    100μlMgSO 4
    160μl胆固醇
    214μltBHP [库存= 70%]因此,终浓度= 15.4 mM

致谢

我们感谢Blackwell和Ewald实验室开发和提炼这些化验。 Nadine Herrmann和Eline Jongsma(ETH Zurich)的图片和电影信用。

参考

  1. An,JH and Blackwell,TK(2003)。&nbsp; SKN -1链接。 elegans mesendodermal规范到保守的氧化应激反应。 Genes Dev 17(15):1882-1893。
  2. Blackwell,TK,Steinbaugh,MJ,Hourihan,JM,Ewald,CY和Isik,M。(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。免费的Radic Biol Med 88“/ =”_ blank“> SKN-1 / Nrf,应激反应和老化在秀丽隐杆线虫 (Pt B):290-301。
  3. Ewald,CY,Hourihan,JM,Bland,MS,Obieglo,C.,Katic,I.,Moronetti Mazzeo,LE,Alcedo,J.,Blackwell,TK and Hynes,NE(2017)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/28085666“target =”_ blank“> NADPH氧化酶介导的氧化还原信号通过记录1促进氧化应激抵抗和寿命在C. elegans 。 Elife 6。
  4. Ewald,CY,Landis,JN,Porter Abate,J.,Murphy,CT and Blackwell,TK(2015)。&nbsp; 519(7541):97-101。
  5. 他,FL(2011)。常见的蠕虫媒体和缓冲区。 Bio-protocol Bio101:&nbsp; e55。
  6. Hourihan,JM,Moronetti Mazzeo,LE,Fernandez-Cardenas,LP和Blackwell,TK(2016)。&nbsp; 半胱氨酸硫基化指导IRE-1以激活SKN-1 / Nrf2抗氧化反应。细胞 63(4):553-566。 br />
  7. Oliveira,RP,Porter Abate,J.,Dilks,K.,Landis,J.,Ashraf,J.,Murphy,CT and Blackwell,TK(2009)。&lt; a class =“ke-insertfile”href = http://www.ncbi.nlm.nih.gov/pubmed/19575768“target =”_ blank“>由秀丽隐杆线虫组成的条件适应性应激和长寿基因调控SKN-1 / Nrf。 a> 老化单元 8(5):524-541。
  8. Robida-Stubbs,S.,Glover-Cutter,K.,Lamming,DW,Mizunuma,M.,Narasimhan,SD,Neumann-Haefelin,E.,Sabatini,DM和Blackwell,TK(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/22560223”target =“_ blank”> TOR信号和雷帕霉素通过调节SKN-1 / Nrf和DAF-16影响寿命/ FoxO。 Cell Metab 15(5):713-724
  9. Steinbaugh,MJ,Narasimhan,SD,Robida-Stubbs,S.,Moronetti Mazzeo,LE,Dreyfuss,JM,Hourihan,JM,Raghavan,P.,Operana,TN,Esmaillie,R。和Blackwell,TK(2015) ; 对SKN-1 / Nrf的脂质介导的调节响应生殖细胞缺失。 4.
  10. Tullet,JM,Hertweck,M.,An,JH,Baker,J.,Hwang,JY,Liu,S.,Oliveira,RP,Baumeister,R.and Blackwell,TK(2008)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/18358814“target =”_ blank“>通过胰岛素样信号直接抑制寿命促进因子SKN-1 >℃。电影。 细胞 132(6):1025-1038。
  11. Wang,J.,Robida-Stubbs,S.,Tullet,JM,Rual,JF,Vidal,M。和Blackwell,TK(2010)。&lt; a class =“ke-insertfile”href =“http: www.ncbi.nlm.nih.gov/pubmed/20700440“target =”_ blank“> RNAi筛选表明SKN-1依赖性转录反应在抗逆性和延缓衰老中的寿命。 PLoS Genet 6(8)。
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Copyright Ewald et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ewald, C. Y., Hourihan, J. M. and Blackwell, T. K. (2017). Oxidative Stress Assays (arsenite and tBHP) in Caenorhabditis elegans. Bio-protocol 7(13): e2365. DOI: 10.21769/BioProtoc.2365.
  2. Ewald, C. Y., Hourihan, J. M., Bland, M. S., Obieglo, C., Katic, I., Moronetti Mazzeo, L. E., Alcedo, J., Blackwell, T. K. and Hynes, N. E. (2017). NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans. Elife 6.
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