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ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent
使用CellROX绿色试剂检测布朗葡萄藻集落中的ROS

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Abstract

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Keywords: Botryococcus braunii(布朗葡萄藻), CellROX Green(CellROX绿色), Fluorescence(荧光), Hydrophobic(疏水), ROS(ROS), Stress(应激), Triton X-100(Triton X-100)

Background

Among the first methods developed to detect and quantify hydrogen peroxide and other organic hydroperoxides was the use of titanium (IV) ion (MacNevin and Urone, 1953). The yellow color resulted from the completion of titanium (IV) and peroxide molecules was detected by colorimetry. This method was used to detect endogenous peroxides, and to assay the catalase activity in two varieties of pear fruits for correlation with fruit ripening (Brennan and Frenkel, 1977). Another method to detect lipid hydroperoxides is based on thiobarbituric acid (TBA) and was used to measure the deterioration of foods such as milk (Sidwell et al., 1955). Although this method did not use organic solvents, steam distillation of an acidified slurry was necessary to detect hydroperoxides, and the resulting red color was quantified spectrophotometrically. The procedures described above have several disadvantages such as low sensitivity, interference with other compounds, and use of solvents or substances which may damage the living cells. A more sensitive method was developed in which the blue fluorescence of scopoletin (6-methyl-7-hydroxy-1:2-benzopyrone) disappeared after its oxidation by peroxidase enzyme (Andreae, 1955; Perschke and Broda, 1961). This method was used to detect the H2O2 production by NADPH in the microsomes from rat liver (Thurman et al., 1972). However, scopoletin is expensive, difficult to extract, and is an extremely toxic natural compound (Ojewole and Adesina, 1983a and 1983b). On the other hand, fluorescein is a dye chemically synthesized (Baeyer, 1871) and the chemical structure was elucidated (Markuszewski and Diehl, 1980). The fluorescence of both compounds, scopoletin and fluorescein, was then explained based on their similar chemical structure. So, further development of novel fluorescent dyes more stable and versatile allowed their use in very specific applications (Cathcart et al., 1983). For instance, 2,7-dichlorohydro-fluorescein diacetate (DCFH-DA) was used to study the intracellular production of active oxygen in the brown alga Fucus evanescens (Collén and Davison, 1997). The same compound DCFH-DA was also used to detect oxidative stress tolerance by abscisic acid (ABA) in the green microalga Chlamydomonas reinhardtii (Yoshida et al., 2003). Due to the wide application of these fluorescent dyes, private companies developed other compounds with different properties and each designed for specific applications. CellROX Green Reagent was designed to detect the production of ROS in living cells. So, we chose this dye to detect ROS in early times in B. braunii living cells (Life Technologies Corp., 2012). These reagents are cell-permeable and show no or very weak fluorescence in a reduced state, but their oxidation results in a strong fluorescence. In presence of ROS, the CellROX Green Reagent undergoes oxidation and produce green fluorescence followed by its binding to the DNA in the nucleus. This fact allows us to distinguish between the fluorescence resulting from ROS and the fluorescence from the chlorophyll molecule. Furthermore, this reagent can be fixed with formaldehyde and is compatible with some detergents. These characteristics of CellROX Green Reagent made it suitable to analyze ROS production in stress conditions in cells of the colonial microalga Botryococcus braunii race B (Nonomura, 1988; Banerjee et al., 2002).

Materials and Reagents

  1. Pipette tips 200 μl (Científica Senna, catalog number: 5-20236 )
  2. 96-well microplate polypropylene (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 267245 )
  3. Glass microscope slide (Corning, catalog number: 2947-75X25 )
  4. Coverslip (Corning, catalog number: 2890-22 )
    Note: This product has been discontinued.
  5. Aluminum foil (Reynolds Wrap 15 m x 30 cm)
  6. CellROX® Green Reagent (Thermo Fischer Scientific, InvitrogenTM, catalog number: C10444 , Excitation/Emission, 485/520 nm)
  7. Triton X-100 (Karal, catalog number: 9015 )
  8. Methyl jasmonate (abbreviated MeJA) (Sigma-Aldrich, catalog number: 392707-5ML )
  9. Potassium nitrate (KNO3) (Karal, catalog number: 5082 )
  10. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Karal, catalog number: 6056 )
  11. Potassium phosphate dibasic (K2HPO4) (Karal, catalog number: 5080 )
  12. Calcium chloride dihydrate (CaCl2·2H2O) (Karal, catalog number: 2016 )
  13. Ethylenediaminetetraacetic acid ferric-sodium salt (Fe·Na·EDTA) (Sigma-Aldrich, catalog number: E6760-100G )
  14. Sulfuric acid (H2SO4) (Karal, catalog number: 1032 )
  15. Boric acid (H3BO4) (Karal, catalog number: 7021 )
  16. Manganese sulfate monohydrate (MnSO4·H2O) (Karal, catalog number: 1069 )
  17. Zinc sulfate monohydrate (ZnSO4·7H2O) (Karal, catalog number: 4089 )
  18. Cupric sulfate pentahydrate (CuSO4·5H2O) (Karal, catalog number: 8024 )
  19. Sodium molybdate dihydrate (NaMoO4·2H2O) (Karal, catalog number: 4072 )
  20. Cobalt(II) sulfate heptahydrate (CoSO4·7H2O) (Sigma-Aldrich, catalog number: 12933 )
    Note: This product has been discontinued.
  21. Sodium chloride (NaCl) (Karal, catalog number: 6052 )
  22. Potassium chloride (KCl) (Karal, catalog number: 5087 )
  23. Sodium bicarbonate (NaHCO3) (Karal, catalog number: 5010 )
  24. Sodium phosphate dibasic (Na2HPO4) (Karal, catalog number: 6005 )
  25. Modified CHU-13 media (see Recipes)
  26. 1x phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. 1.5 L flask (Corning, PYREX®, catalog number: 4980-1XL )
  2. Micropipettes (Mettler-Toledo International, Rainin®, catalog numbers: 17014392 , 17014382 and 17011790 )
  3. Incubator shaker (Select BioProducts, model: IncuMixTM Incubator Shaker, catalog number: SBS256 )
  4. Centrifuge (Labnet International, model: SpectrafugeTM 16M, catalog number: C0160 )
  5. Optical microscope (ZEISS, model: Axio Lab.A1 ) equipped with 470 nm LED module used for fluorochrome excitation and a set of 38 Endow GFP Filters (free exchange (E) EX BP 470/40, BS FT 495, EM BP 525/50) to detect the emission of the fluorochrome
  6. Digital camera (ZEISS, model: AxioCam ICc3 Rev.3 )
  7. pH meter (Cole-Parmer, Jenway, model: 3510 )
  8. Autoclave Sterilmatic (Market Forge Industries, model: STM-EL )

Software

  1. ZEN lite 2011 (ZEISS)
  2. GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com)

Procedure

  1. Harvest samples of the algal culture grown in modified Chu-13 media (see Recipes), at different time points after specific stress treatments. There should be sufficient colonies in 100 μl to be clearly observed under a microscope. If your alga can be sedimented after centrifugation, 100 μl should make at least a 20 μl of pellet. If the alga doesn’t sediment as in the case of B. braunii, which floats because of the surrounding hydrocarbons, then the OD at 600 nm should give at least 0.5. If needed, concentrate the cells by centrifugation, filtration, or other methods that are suitable for your sample.
  2. Transfer 100 μl of each sample in a 96-well microplate and mix with 2 μl of 5 mM CellROX Green, incubate the microplate for 30 min at 36 °C in the dark by shaking at 120 rpm in an incubator shaker.
  3. Then incline the plates, carefully discard the liquid with a micropipette and wash the cells twice with 100 μl of 1x PBS (see Recipes) containing 0.1% Triton X-100, by shaking at 120 rpm for 5 min at room temperature. Triton X-100 is a commonly used detergent in laboratories widely used to permeabilize the membranes of living cells.
  4. Transfer an aliquot of 10-20 μl of each sample onto a glass microscope slide, cover with a coverslip, and observe under the microscope.
  5. Observe samples first under the white light to locate the algal colonies and then switch to fluorescence conditions. Count at least 100 colonies for each sample.
  6. Colonies with more that 90% of cells having the fluorescent nuclei are considered ROS positive.
  7. Take pictures with a digital camera and the representative images are shown below (Figure 1).


    Figure 1. Staining of Botryococcus braunii with CellROX dye. Detect ROS in vivo by analyzing fluorescent nuclei of B. braunii cells in each colony. Upper panels are under white light and lower panels are under fluorescent conditions. A. Control without stress treatment; B. Treatment with strong stress inducer (10 μM MeJA for 60 min); C. Treatment with weak stress inducer (120 mM NaHCO3, for 60 min).

  8. Determine the percentage of ROS positive colonies according to the equation:

    (FC/TC) x 100 = %PC

    where, FC = number of colonies with fluorescence, TC = total number of observed colonies, and PC = percent of ROS positive colonies.

Data analysis

With this procedure both the concentration of the inducers and the time of induction are optimized. The number of ROS positive colonies after CellROX staining is determined by counting the fluorescent cells induced with the different treatments for 10, 60 and 120 min. Data represent the mean ± SE of at least three replicates. The statistical significance is evaluated with the Tukey’s test (P value = ** 0.0022, *** 0.0003, **** < 0.0001). Statistical tests are performed using GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com). Data are visualized using Microsoft Excel for Mac 2011 version 14.1.0. The number of positive colonies for ROS are expressed in % positive colonies (Table 1).

Table 1. Percentage of ROS positive B. braunii colonies. Take samples at different times and with different concentrations of the inducer.


These data show that the best conditions to detect ROS-positive colonies of B. braunii, were 60 min for 10 μM MeJA as well as for 120 mM NaHCO3. However, the best conditions regarding time and concentration of inducers must be determined for other microalgae.

Notes

  1. A small number of colonies are identified as ROS positive even without treatment of any stress inducer, this may be due to damage during handling of samples. However, they never exceed 13.5% of the total colonies analyzed and are lower than the number of colonies observed in any of the treatments tested.
  2. Due to the colony organization of B. braunii it was sometimes difficult to count individual cells in a selected sample without moving the field under the microscope. So, once the field was fixed under fluorescent light, colonies were considered ROS positive if more than 90% of the cells in the field had fluorescent nuclei. It is recommended to try different focus on the same colony to distinguish the stained nuclei of the cells at different planes.
  3. Triton X-100 was added at the washing steps to improve the introduction of the CellROX Green into the B. braunii cells. In step 2 of the Procedure, when the dye was added, it covered and bonded to the surface of the colony but hardly got into it to act in the inner cells. In step 3, the Triton X-100 allowed the introduction of the dye by its detergent property. This was a key step because of this alga secretes large amounts of liquid hydrocarbons which surround the cells, creating a highly hydrophobic extracellular environment. During the washing steps, the dye reached the inner cells to detect ROS and the excess of CellROX was discarded. In our hands, after the second wash, the reaction stopped because all produced ROS were detected with the dye. Perhaps, other microalgae do not require this step although it may help to get better results, mainly if the cultures were forced to produce a high amount of lipids.

Recipes

  1. Modified CHU-13 media
    0.4 g/L KNO3
    0.1 g/L MgSO4·7H2O
    0.052 g/L K2HPO4
    0.054 g/L CaCl2·2H2O
    0.01 g/L Fe·Na·EDTA
    5 ml/L trace elements
    Dissolve each salt in 800 ml deionized water. Set the pH to 7.2-7.5 using diluted H2SO4. The pH of the media should be over 7.0, do not adjust with NaOH. In case the pH is below 7.0, discard the solution and prepare it again
    Trace elements:
    572 mg/L H3BO4
    308 mg/L MnSO4·H2O
    44 mg/L ZnSO4·7H2O
    16 mg/L CuSO4·5H2O
    12 mg/L NaMoO4·2H2O
    18 mg/L CoSO4·7H2O
    Mix all in 900 ml deionized water in a 1.5 L flask, cover with aluminum foil and sterilize by autoclaving at 121 °C for 20 min
  2. 1x phosphate-buffered saline (PBS)
    8 g/L NaCl
    0.2 g/L KCl
    1.44 g/L Na2HPO4
    0.24 g/L KH2PO4
    Dissolve the reagents in 800 ml of deionized water. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Sterilize by autoclaving for 20 min at 121 °C or by filter sterilization. Store PBS at room temperature

Acknowledgments

This technique was adapted from the original procedure described in the Manual of CellROX® Oxidative Stress Reagents Man0003555 from Life Technologies Corp. This work was supported by a PhD scholarship to IC-C from Consejo Nacional de Ciencia y Tecnología (CONACYT) Mexico, and grant from the 2012 Texas A&M University CONACYT Collaborative Research Grant Program to EL-G and TPD.

References

  1. Andreae, W. A. (1955). A sensitive method for the estimation of hydrogen peroxide in biological materials. Nature 175(4463): 859-860.
  2. Baeyer, A. (1871). Ueber eine neue Klasse von Farbstoffen. Eur J Inorg Chemy 4(2): 555-558.
  3. Banerjee, A., Sharma, R., Chisti, Y. and Banerjee, U. C. (2002). Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 22(3): 245–279.
  4. Brennan, T. and Frenkel, C. (1977). Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol 59(3): 411-416.
  5. Cathcart, R., Schwiers, E. and Ames, B. N. (1983). Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem 134(1): 111-116.
  6. Collén, J. and Davison, I. R. (1997). In vivo measurement of active oxygen production in the brown alga Fucus evanescens using 2’,7’-dichlorohydrofluorescein diacetate. J Phycol 33(4): 643-648.
  7. Life Technologies Corp. (2012). Manual of CellROX® Oxidative Stress Reagents. Man0003555: 1-6.
  8. MacNevin, W. M. and Urone, P. F. (1953). Separation of hydrogen peroxide from organic hydroperoxides. Anal Chem 25(11): 1760-1761.
  9. Markuszewski, R. and Diehl, H. (1980). The infrared spectra and structures of the three solid forms of fluorescein and related compounds. Talanta 27(11): 937-946.
  10. Nonomura, A. M. (1988). Botryococcus braunii var. showa (Chlorophyceae) from Berkeley, California, United States of America. Japanese J Phycol 36: 285-291.
  11. Ojewole, J. A. and Adesina, S. K. (1983a). Cardiovascular and neuromuscular actions of scopoletin from fruit of Tetrapleura tetraptera. Planta Med 49(2): 99-102.
  12. Ojewole, J. A. O. and Adesina, S. K. (1983b). Mechanism of the hypotensive effect of scopoletin isolated from the fruit of Tetrapleura tetraptera. Planta Med 49(9): 46-50.
  13. Perschke, H. and Broda, E. (1961). Determination of very small amounts of hydrogen peroxide. Nature 190: 257-258.
  14. Sidwell, C. G., Salwin, H. and Mitchell, J. H. (1955). Measurement of oxidation in dried milk products with thiobarbituric acid. J Am Oil Chem Soc 32(1): 13-16.
  15. Thurman, R. G., Ley, H. G. and Scholz, R. (1972). Hepatic microsomal ethanol oxidation. Hydrogen peroxide formation and the role of catalase. Eur J Biochem 25(3): 420-430.
  16. Yoshida, K., Igarashi, E., Mukai, M., Hirata, K. and Miyamoto, K. (2003). Induction of tolerance to oxidative stress in the green alga, Chlamydomonas reinhardtii, by abscisic acid. Plant Cell Environ 26(3): 451-457.

简介

我们分析了响应于几种胁迫诱导剂如NaCl,NaHCO 3,水杨酸(SA),茉莉酸甲酯和乙酸的菌落形成绿色微藻葡萄球菌中的活性氧(ROS)积累。 使用使用荧光染料CellROX Green的染色测定。 CellROX Green是用于测量活细胞氧化应激的荧光探针。 该染料在细胞内处于弱的荧光,但是在通过ROS氧化并随后与DNA结合时呈现出明亮的绿色光稳定荧光。 由布鲁氏杆菌生产和排泄的大量液体烃产生了高度疏水的细胞外环境,使得难以在该微藻上研究短时间的防御反应。 这里开发的程序允许我们在用不同的应激诱导剂处理细胞后,在短时间内(以分钟为单位),在这种微藻中检测ROS。
【背景】用于检测和定量过氧化氢和其他有机氢过氧化物的第一种方法是使用钛(IV)离子(MacNevin和Urone,1953)。由于钛(IV)的完成导致黄色,通过比色法检测过氧化物分子。该方法用于检测内源性过氧化物,并测定两种梨果实中的过氧化氢酶活性与果实成熟相关(Brennan和Frenkel,1977)。检测脂质氢过氧化物的另一种方法是基于硫代巴比妥酸(TBA),并用于测量诸如牛奶等食品的变质(Sidwell等,1955)。虽然该方法不使用有机溶剂,但是需要对酸化浆料进行蒸汽蒸馏来检测氢过氧化物,并且通过分光光度法定量得到的红色。上述方法具有诸如低灵敏度,与其它化合物的干扰以及可能损坏活细胞的溶剂或物质的使用等缺点。开发了一种更灵敏的方法,其中scopoletin(6-甲基-7-羟基-1,2-苯并吡喃酮)的蓝色荧光在其被过氧化物酶氧化后消失(Andreae,1955; Perschke和Broda,1961)。该方法用于检测来自大鼠肝脏的微粒体中NADPH的H2O2产生(Thurman等,1972)。然而,scopoletin是昂贵的,难以提取,并且是非常有毒的天然化合物(Ojewole和Adesina,1983a和1983b)。另一方面,荧光素是化学合成的染料(Baeyer,1871),化学结构被阐明(Markuszewski和Diehl,1980)。然后基于它们的类似化学结构来解释两种化合物,scopoletin和荧光素的荧光。因此,进一步开发更稳定和多用途的新型荧光染料可以在非常具体的应用中使用(Cathcart等,1983)。例如,使用2,7-二氯氢化荧光素二乙酸酯(DCFH-DA)来研究褐藻(Fucus evanescens)中活性氧的细胞内产生(Collén和Davison,1997)。同样的化合物DCFH-DA也用于检测绿色微藻衣藻中脱落酸(ABA)的氧化应激耐受性(Yoshida等,2003)。由于这些荧光染料的广泛应用,私营公司开发出具有不同性能的其他化合物,并且每个都针对特定应用设计。 CellROX Green Reagent旨在检测活细胞中ROS的产生。因此,我们选择这种染料早期在布鲁氏菌活细胞中检测ROS(Life Technologies Corp.,2012)。这些试剂是细胞可渗透的,并且在还原状态下不显示或非常弱的荧光,但是它们的氧化导致强荧光。在ROS存在下,CellROX Green试剂经历氧化并产生绿色荧光,然后与核中的DNA结合。这个事实使我们能够区分由ROS产生的荧光和来自叶绿素分子的荧光。此外,该试剂可以用甲醛固定,并与一些洗涤剂相容。 CellROX Green Reagent的这些特征使其适用于分析殖民地微藻Botryococcus braunii种族B细胞的应激条件下的ROS产生(Nonomura,1988; Banerjee et al。,2002)。

关键字:布朗葡萄藻, CellROX绿色, 荧光, 疏水, ROS, 应激, Triton X-100

材料和试剂

  1. 移液瓶提示200μl(CientíficaSenna,目录号:5-20236)
  2. 96孔微板聚丙烯(Thermo Fischer Scientific,Thermo Scientific TM,目录号:267245)
  3. 玻璃显微镜幻灯片(康宁,目录号:2947-75X25)
  4. Coverslip(康宁,目录号:2890-22)
    注意:本产品已停产。
  5. 铝箔(雷诺缠绕15米×30厘米)
  6. CellROX ®绿色试剂(Thermo Fischer Scientific,Invitrogen TM,目录号:C10444,激发/发射,485/520nm)
  7. Triton X-100(Karal,目录号:9015)
  8. 茉莉酸甲酯(缩写为MeJA)(Sigma-Aldrich,目录号:392707-5ML)
  9. 硝酸钾(KNO 3)(Karal,目录号:5082)
  10. 七水硫酸镁(MgSO 4·7H 2 O)(Karal,目录号:6056)
  11. 磷酸氢二钾(K 2 H 2 HPO 4)(Karal,目录号:5080)
  12. 氯化钙二水合物(CaCl 2·2H 2 O)(Karal,目录号:2016)
  13. 乙二胺四乙酸铁钠盐(Fe·Na·EDTA)(Sigma-Aldrich,目录号:E6760-100G)
  14. 硫酸(H 2 SO 3 SO 4)(Karal,目录号:1032)
  15. 硼酸(H 3 3 BO 4)(Karal,目录号:7021)
  16. 硫酸锰一水合物(MnSO 4·H 2 O)(Karal,目录号:1069)
  17. 硫酸锌一水合物(ZnSO 4·7H 2 O)(Karal,目录号:4089)
  18. 五水合硫酸铜(CuSO 4·5H 2 O)(Karal,目录号:8024)
  19. 钼酸钠二水合物(NaMoO 4·2H 2 O)(Karal,目录号:4072)
  20. 硫酸钴(II)七水合物(CoSO 4·7H 2 O)(Sigma-Aldrich,目录号:12933)
    注意:本产品已停产。
  21. 氯化钠(NaCl)(Karal,目录号:6052)
  22. 氯化钾(KCl)(Karal,目录号:5087)
  23. 碳酸氢钠(NaHCO 3)(Karal,目录号:5010)
  24. 磷酸氢二钠(Na 2 HPO 4)(Karal,目录号:6005)
  25. 修改的CHU-13介质(见配方)
  26. 1x磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 1.5升烧瓶(Corning,PYREX ,目录号:4980-1XL)
  2. 微量移液器(Mettler-Toledo International,Rainin ®,目录号:17014392,17014382和17011790)
  3. 培养箱振荡器(选择BioProducts,型号:IncuMix TM培养箱振荡器,目录号:SBS256)
  4. 离心机(Labnet International,型号:Spectrafuge TM 16M,目录号:C0160)
  5. 配备了用于荧光染料激发的470nm LED模块的光学显微镜(ZEISS,型号:Axio Lab.A1)和一组38个Endow GFP过滤器(自由交换(E)EX BP 470/40,BS FT 495,EM BP 525 / 50)以检测荧光染料的排放
  6. 数码相机(ZEISS,型号:AxioCam ICc3 Rev.3)
  7. pH计(Cole-Parmer,Jenway,型号:3510)
  8. 高压灭菌器(市场锻造行业,型号:STM-EL)

软件

  1. ZEN lite 2011(ZEISS)
  2. GraphPad Prism版本6.00适用于Mac OS X,GraphPad Software,La Jolla California USA( http:// www .graphpad.com

程序

  1. 在特定压力处理后的不同时间点收获在改良的Chu-13培养基(见食谱)中生长的藻类培养物样品。在显微镜下应该清楚地观察到100μl中足够的菌落。如果您的藻类在离心后可以沉淀,100μl应至少制成20μl的颗粒。如果藻类不像B那样沉淀。 braunii ,由于周围的碳氢化合物而漂浮,则600nm处的OD应至少为0.5。如果需要,通过离心,过滤或其他适合您样品的方法浓缩细胞。
  2. 在96孔微量培养板中转移100μl各样品,并与2μl5mM CellROX Green混合,在37℃下,在培养箱中以120rpm摇动,在黑暗中孵育30分钟。
  3. 然后倾斜板,用微量移液管小心地丢弃液体,并在室温下以120rpm摇动5分钟,用含有0.1%Triton X-100的100μl1x PBS(参见食谱)洗涤细胞两次。 Triton X-100是广泛用于透过活细胞膜的实验室中常用的洗涤剂。
  4. 将10-20μl每个样品的等分试样转移到玻璃显微镜载玻片上,盖上盖玻片,并在显微镜下观察。
  5. 首先在白光下观察样品以定位藻类菌落,然后切换到荧光条件。每个样本计数至少100个菌落。
  6. 具有更多90%具有荧光核的细胞的殖民地被认为是ROS阳性
  7. 使用数码相机拍照,代表图像如下图所示(图1)

    图1.用CellROX染料染色乳香葡萄球菌。通过分析B的荧光核,检测体内的ROS 。 braunii 细胞在每个殖民地。上面板在白光下,下面板处于荧光条件下。控制无压力处理; B.用强应力诱导剂治疗(10μMMeJA 60分钟); C.用弱应力诱导剂(120mM NaHCO 3,60分钟)处理。

  8. 根据以下方程确定ROS阳性菌落的百分比:

    (FC / TC)x 100 =%PC

    其中,FC =具有荧光的菌落数,TC =观察到的菌落总数,PC = ROS阳性菌落的百分比。

数据分析

通过该方法,诱导剂的浓度和诱导时间都被优化。通过计数用不同处理诱导的荧光细胞10,60和120分钟来确定CellROX染色后的ROS阳性菌落数。数据表示至少三次重复的平均值±SE。统计显着性用Tukey's检验(P 值= ** 0.0022,*** 0.0003,**** <0.0001)进行评估。统计测试使用GraphPad Prism版本6.00 for Mac OS X,GraphPad Software,La Jolla California USA执行( http://www.graphpad.com )。使用Microsoft Excel for Mac 2011版本14.1.0可视化数据。 ROS的阳性菌落数以%阳性菌落表示(表1)
表1. ROS阳性百分比B。 Braunii 菌落。在不同时间和不同浓度的诱导物中取样。


这些数据表明,对于10μMMeJA以及120mM NaHCO 3的检测,检测胸膜布鲁氏杆菌的ROS阳性菌落的最佳条件为60分钟。然而,关于诱导剂的时间和浓度的最佳条件必须对其他微藻确定。

笔记

  1. 即使没有治疗任何应激诱导剂,也将少量菌落鉴定为ROS阳性,这可能是由于在处理样品期间的损害。然而,它们不超过分析的总菌落的13.5%,并且低于在任何所测试的处理中观察到的菌落数。
  2. 由于B的殖民地组织。 braunii 有时难以在选定样品中计数单个细胞,而不在显微镜下移动场。因此,一旦野外在荧光灯下固定,如果现场超过90%的细胞具有荧光核,则将菌落视为ROS阳性。建议尝试不同的重点在同一个殖民地区分细胞在不同的平面染色的核
  3. 在洗涤步骤中加入Triton X-100,以改进将CellROX Green引入到B中。胸罩细胞。在该步骤的步骤2中,当添加染料时,其覆盖并结合到菌落的表面,但很难进入其内部细胞。在步骤3中,Triton X-100允许通过其洗涤剂性质引入染料。这是一个关键步骤,因为这个藻类分泌了大量围绕细胞的液体烃,产生了高度疏水的细胞外环境。在洗涤步骤中,染料到达内细胞以检测ROS,并且丢弃多余的CellROX。在我们手中,第二次洗涤后,反应停止,因为用染料检测到所有产生的ROS。也许,其他微藻不需要这一步,虽然它可能有助于获得更好的结果,主要是如果文化被迫产生大量的脂质。

食谱

  1. 修改CHU-13媒体
    0.4g / L KNO 3
    0.1g / L MgSO 4·7H 2 O→/ / 0.052g / L K 2 HPO 4
    0.054g / L CaCl 2·2H 2 O
    0.01g / L Fe·Na·EDTA
    5 ml / L痕量元素
    将每个盐溶解在800ml去离子水中。使用稀释的H 2 SO 4将pH设定为7.2-7.5。介质的pH应超过7.0,不能用NaOH调节。如果pH低于7.0,请丢弃溶液并再次准备 微量元素:
    572mg / L H 3 BO 4
    308mg / L MnSO 4·H 2 O
    44mg / L ZnSO 4·7H 2 O
    16mg / L CuSO 4·5H 2 O
    12mg / L NaMoO 4 / 2H 2 O 18mg / L CoSO 4·7H 2 O
    在1.5升烧瓶中将900ml去离子水全部混合,用铝箔覆盖,并在121℃高压灭菌20分钟消毒。
  2. 1x磷酸盐缓冲盐水(PBS)
    8克/升NaCl
    0.2克/升KCl
    1.44g / L Na 2 HPO 4
    0.24g / L KH 2 PO 4
    将试剂溶于800毫升去离子水中。用HCl调节pH至7.4(或7.2,如果需要),然后将H 2 O加至1L。在121℃下通过高压灭菌20分钟灭菌或通过过滤灭菌灭菌。在室温下储存PBS

致谢

该技术根据Life Technologies Corp.的CellROX Oxidative Stress Reagents Man0003555手册中描述的原始程序改编。该工作得到了Consejo Nacional de Ciencia yTecnología的IC-C博士学位奖学金的支持(CONACYT)墨西哥,并从德克萨斯A&amp; M大学CONACYT合作研究授权计划授予EL-G和TPD。

参考

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  3. Banerjee,A.,Sharma,R.,Chisti,Y.,and Banerjee,UC(2002)。 Botryococcus braunii :碳氢化合物和其他化学物质的可再生资源 Crit Rev Biotechnol 22(3):245 -279。
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  5. Cathcart,R.,Schwiers,E.和Ames,BN(1983)。&nbsp; 使用荧光二氯荧光素检测检测皮摩尔水平的氢过氧化物。 Anal Biochem 134(1):111-116。
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  9. Markuszewski,R.和Diehl,H。(1980)。三种固体形式的荧光素和相关化合物的红外光谱和结构。 Talanta 27(11):937-946。
  10. Nonomura,AM(1988)。&nbsp; Botryococcus braunii 变种美国加利福尼亚州伯克利市的showa(Chlorophyceae)。日本J Phycol 36:285-291。
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引用:Lozoya-Gloria, E., Cornejo-Corona, I., Thapa, H. R., Browne, D. R. and Devarenne, T. P. (2017). ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent. Bio-protocol 7(16): e2508. DOI: 10.21769/BioProtoc.2508.
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