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[Bio101] MTT Assay for Cytotoxicity Assessment in Oryza sativa Root Tissue
[Bio101] MTT法评估亚洲水稻根系组织中的细胞毒性   

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Abstract

Cytotoxicity of different compounds are commonly evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay is mainly used to study cell viability in cell lines (Carmichael et al., 1987). In this study, the protocol is being used to determine the cell viability of plant roots, treated with different stress inducing agents. The basis of the assay is that the dye enters the living cell’s mitochondrion where it is reduced to insoluble formazan, which is solubilised by directly treating the cells with organic solvent (DMSO). Intensity of colour is directly proportional to the amount of formazan produced.

In the present study, plants were treated for 16 h, with several phytotoxic agents, then the roots were incubated in MTT solution for 4 h. To solubilise the formazan, roots were excised. 2 N potassium hydroxide (KOH) along with DMSO was used to solubilize the cell wall components and thereby liberating the formazan granules in the DMSO solution. The rate of the cell viability was measured by measuring the colour intensity of the formazan.

Keywords: Cell viability(细胞成活力), Cytotoxicity(细胞毒性), MTT assay(MTT法), Plant tissue(植物组织), Rice seedling(水稻秧苗)

Background

This protocol is designed to determine plant cell viability directly from root tissue. Till date, MTT assay has been profusely used for mammalian cell proliferation and viability assay. In case of plants usually the 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay (Kaundal et al., 2012) is preferably used. Cost effective MTT assay can also be used for plant tissue viability assay instead of XTT assay. This protocol can be used to determine the cytotoxicity of different stress inducing agents and their IC50 doses (the concentration of phytotoxic agent at which 50% cell death is obtained). Nicotinamide adenine dinucleotide phosphate (NAD(P)H) dependent dehydrogenase enzyme present in mitochondrion of living cell is capable of reducing tetrazolium dye MTT 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenyltetrazolium bromide to an insoluble purple coloured formazan, which is measured spectrophotometrically. This reduction takes place when the mitochondrial enzymes are active and hence the degree of reduction can be directly correlated with the number of viable cells. The metabolically inactive cells will not show this property.

Materials and Reagents

  1. Culture tubes (Borosil, catalog number: 9910010 )
  2. Tissue paper (Kim wipes) (Tarsons, catalog number: 370080 )
  3. 2 ml microcentrifuge tube (gem. gov. in, Genaxy, catalog number: GEN-MT-200-C )
  4. Aluminium foil FreshwrappR (Hindalco)
  5. Petri plates (Borosil, catalog number: 3160065 )
  6. Rice Oryza sativa cv. IR64 (6 days old) (Rice Research Station Government of West Bengal, Chinsurah R.S., Hooghly, India)
  7. Stress inducing agents:
    1. Nanoscale zero-valent iron nanoparticles (nZVI) (gifted by Prof. A. Mukherjee, Vellore Institute of Technology, Tamil Nadu, India)
    2. Cadmium chloride monohydrate (CdCl2·H2O) (Merck, catalog number: 61813101001730 )
    3. Sodium chloride (NaCl) (HiMedia Laboratories, catalog number: MB023-500G )
    4. Mannitol(C6H14O6) (Sisco Research Laboratories, catalog number: 134889 )
  8. Potassium hydroxide (KOH) (HiMedia Laboratories, catalog number: GRM251-500G )
  9. 99.99% dimethyl sulfoxide (DMSO) (Merck, catalog number: 1.07046.0521 )
  10. Antifungal agent: DithaneR M-45 75% WP (Dow AgroSciences)
  11. Sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O) (Merck, CAS number: 200-664-3)
  12. Di-sodium hydrogen phosphate anhydrous (Na2HPO4) (Merck, CAS number: 231-448-7)
  13. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) (Sisco Research Laboratories, catalog number: 33611 )
  14. 70% alcohol (Analytical Z Reagent, Hong Young Chemicals, catalog number: 15005-51 )
  15. Deionised water
  16. 50 mM sodium phosphate buffer (pH 7) (see Recipes)
  17. 1x Hoagland’s solution (see Recipes)
    1. Potassium dihydrogen phosphate (KH2PO4) (Sisco Research Laboratories, CAS number: 7778-77-0)
    2. Potassium nitrate (KNO3) (Merck, CAS number: 231-818-8)
    3. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (Merck, CAS number: 233-332-1)
    4. Magnesium sulphate heptahydrate (MgSO4·7H2O) (Sisco Research Laboratories, catalog number: 85611 )
    5. Manganese sulphate monohydrate (MnSO4·H2O) (Sisco Research Laboratories, catalog number: 12386 )
    6. Zinc suphate heptahydrate (ZnSO4·7H2O) (Sisco Research Laboratories, catalog number: 75738 )
    7. Copper sulphate pentahydrate (CuSO4·5H2O) (Merck, CAS number: 231-847-6)
    8. Ammonium molybdate monohydrate ((NH4)6Mo7O24·H2O) (Sisco Research Laboratories, catalog number: 69429 )
    9. Boric acid (H3BO3) (Sisco Research Laboratories, MDL number: 22311 )
    10. Ferric chloride (FeCl3) (Thermo Fisher Scientific, catalog number: MFCD00011005 )
  18. Stress inducing agents(see Recipes)

Equipment

  1. Measuring cylinder (Tarsons, catalog numbers: 345040 , 345070 )
  2. Weighing balance (Wensar, model: PGB 610 )
  3. pH meter (Global Electronics, model: DPH 500 )
  4. UV-VIS spectrophotometer (Techcomp, model: UH5300 )
  5. Centrifuge (Eppendorf, model: 5810R , catalog number: 3334)

Procedure

  1. Sterilize the Oryza sativa seeds with 0.2% dithane-M45 and wash thoroughly with deionised water for at least 10-15 times till there is no pungent odour of dithane-M45.
  2. Leave them for 24 h in deionised water for water imbibition.
  3. Germinate the seeds in the dark for 72 h at 30 °C.
  4. Grow seedlings in culture tubes at 30 °C with a photoperiod of 16 h light and 8 h dark for 6 days in nutrient solution (1x Hoagland’s solution).
  5. Add 5 ml of different stress inducing agents (see Recipes) directly to the culture tubes containing seedlings. Add 5 ml of deionised water to the control set, stir the treatments by occasional aeration through bubbling.
  6. Keep the control and treated sets for 16 h.
  7. Remove the seedlings from the growth solution, wipe off till dry with tissue paper.
  8. Weigh ~10 mg of fresh root tissue and transfer to a fresh 2 ml microcentrifuge tube.
  9. Add 1.5 ml of MTT solution (0.25 mg/L, see Recipes) and keep the tubes in the dark for 4 h.
  10. Discard the MTT solution, take the root samples into clean Petriplates without any further washing, cut the roots into 1-2 mm pieces with sterile scalpel (to ensure leaching out of the formed formazan). Add 0.5 ml KOH (2 N) to this and transfer the cut pieces along with KOH solution to 2 ml microcentrifuge tubes.
  11. Add 0.5 ml 99.99% DMSO solution to each tube, to make the final volume 1 ml. The colour of the solution appears as shown in Figure 1.
  12. Centrifuge the tubes briefly at 500 x g for 5 min at room temperature so that the root pieces settle down.
  13. Transfer the clear supernatant into fresh tubes.
  14. Take spectrophotometric readings of the supernatants at 570 nm immediately. Keep the samples in the dark all the time due to photosensitivity of formazan formed. (see Notes 1 and 2)
  15. The O.D. values obtained are for 10 mg tissue, calculate the O.D. value for 1 mg. From the O.D. value determine the percentage of cell death using below-mentioned formula,



    Hence, percentage of cell viability/mg fresh tissue= 100 - percentage of cell death

    Percentage of cell death is shown in Table 1 and Figure 2.


    Figure 1.Pictorial representation of different stages of the MTT assay. Bl-Blank, Co-Control, M-treated with 200 mM mannitol, Na-treated with 200 mM sodium chloride, nZ-treated with 200 mg/L nZVI, Cd-treated with 100 mM cadmium chloride. A. Seedlings being grown in growth chamber; B. Seedlings after treatment with stress inducing agents for 16 h; C. Roots before MTT assay; D. Roots after MTT treatment for 4 h. Co (i), Cd (i), M, Na, nZ, Co (ii), Cd (ii) Roots chopped to 1-2 mm in length. E. Colour intensity of the formazan after solubilized in DMSO.

    Table 1. O.D. value and percentage of cell death



    Figure 2. The percentage of cell death of the root tissue is determined from the OD value at 570 nm. The cell death percentage is maximum for 100 mM CdCl2 followed by 200 mg Nzvi > 200 mM NaCl > 200 mM mannitol.

Notes

  1. Avoid direct light after addition of MTT as it is photosensitive.
  2. Record the absorbance immediately after solubilizing the formazan in DMSO as the colour intensity may change and give erroneous readings.

Recipes

  1. 0.2% dithane solution
    Dissolve 200 mg of dithane in 100 ml autoclaved deionised water
  2. 50 mM sodium phosphate buffer (pH 7)
    1. Prepare 50 mM sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) (Merck) by dissolving 0.78 g of sodium dihydrogen phosphate in 100 ml deionised water
    2. Prepare 50 mM di-sodium hydrogen phosphate anhydrous (Na2HPO4) (Merck) by dissolving 0.7 g of di-sodium hydrogen phosphate in 100 ml deionised water
    3. 55 ml of 50 mM sodium dihydrogen phosphate dihydrate and 45 ml of 50 mM di-sodium hydrogen phosphate anhydrous are mixed to make 50 mM sodium phosphate buffer and adjust the pH to 7
  3. MTT dye
    Prepare stock solution of MTT (10 mg/ml) by dissolving the MTT in 50 mM phosphate buffer (pH 7)
  4. 2 N potassium hydroxide solution
    Dissolve 11.2 g potassium hydroxide in 100 ml deionised water
  5. 1x Hoagland’s solution
    Macronutrients:
    1 mM potassium dihydrogen phosphate (KH2PO4)
    5 mM potassium nitrate (KNO3)
    5 mM Calcium nitrate Ca(NO3)2
    Micronutrients:
    11.8 µM manganese sulphate monohydrate (MnSO4·H2O)
    0.70 µM zinc suphate heptahydrate (ZnSO4·7H2O)
    0.32 µM copper sulphatepentahydrate (CuSO4·5H2O)
    0.16 µM ammonium molybdate monohydrate ((NH4)6Mo7O24·H2O)
    46.3 µM boric acid (H3BO3)
    5 µM ferric chloride (FeCl3)
    Make up to 1 L with autoclaved deionised water
    Adjust the pH to 5.8
  6. Stress inducing agents
    200 mM mannitol
    200 mg/L nZVI
    100 mM CdCl2
    200 mM NaCl
    Dissolved in deionised water

Acknowledgments

Authors wish to acknowledge the Department of Botany, Centre of Advanced Studies, Phase-VII, University of Calcutta for the instrumentation facilities. SM and TG wish to acknowledge DBT, GOI and CSIR for the financial assistance. The authors declare the absence of any conflict of interests.

References

  1. Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D. and Mitchell, J. B. (1987). Evaluation of a tetrazolium-based semi automated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47(4): 936-942.
  2. Kaundal, A., Rojas, C. M. and Mysore, K. S. (2012). Measurement of NADPH oxidase activity in plants. Bio Protoc 2(20): e278.

简介

通常通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT)测定评估不同化合物的细胞毒性。该测定主要用于研究细胞系中的细胞活力(Carmichael等人,1987)。在这项研究中,该协议正在被用来确定植物根的细胞活力,用不同的应激诱导剂处理。该检测的基础是染料进入活细胞的线粒体,然后被还原成不溶性的甲,,通过直接用有机溶剂(DMSO)处理细胞来溶解甲an。颜色强度与产生的甲amount量成正比。

在本研究中,植物处理16小时,用几种植物毒性剂,然后将根在MTT溶液中温育4小时。为了溶解甲,,将根切除。用2N氢氧化钾(KOH)与DMSO一起溶解细胞壁组分,从而释放DMSO溶液中的甲granules颗粒。通过测量甲color的颜色强度来测量细胞存活率。

【背景】该协议旨在直接从根组织中确定植物细胞活力。迄今为止,MTT测定已被广泛用于哺乳动物细胞增殖和活力测定。在植物的情况下,通常使用2,3-双 - (2-甲氧基-4-硝基-5-磺苯基)-2H-四唑-5-甲酰苯胺(XTT)测定法(Kaundal等人, 2012)。成本效益的MTT分析也可以用于植物组织活力测定而不是XTT测定。该方案可用于确定不同应激诱导剂和其IC 50剂量(获得50%细胞死亡的植物毒素剂的浓度)的细胞毒性。存在于活细胞线粒体中的烟酰胺腺嘌呤二核苷酸磷酸(NAD(P)H)依赖性脱氢酶能够将四唑鎓染料MTT3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物还原成用分光光度法测定不溶性紫色甲。当线粒体酶活性降低时,发生这种降低,因此还原程度可以与活细胞的数量直接相关。代谢惰性的细胞不会显示这种性质。

关键字:细胞成活力, 细胞毒性, MTT法, 植物组织, 水稻秧苗

材料和试剂

  1. 培养管(Borosil,目录号:9910010)
  2. 纸巾(Kim wipes)(Tarsons,目录号:370080)
  3. 2ml微量离心管(gem.gov。in,Genaxy,目录号:GEN-MT-200-C)
  4. 铝箔Freshwrapp R(Hindalco)
  5. 培养皿(Borosil,目录号:3160065)
  6. 水稻(Oryza sativa) cv。 IR64(6日龄)(西孟加拉邦政府水稻研究站,印度Hooghly Chinsurah R.S.)
  7. 应激诱导剂:
    1. 纳米级零价铁纳米粒子(nZVI)(由印度泰米尔纳德邦Vellore技术研究所A. Mukherjee教授提供)
    2. 氯化镉一水合物(CdCl 2•2H 2 O)(Merck,MA8M573303)
    3. 氯化钠(NaCl)(HiMedia Laboratories,目录编号:MB023-500G)
    4. 甘露醇(C 6 H 14 O 6)(Sisco Research Laboratories,目录号:134889)
  8. 氢氧化钾(KOH)(HiMedia实验室,目录号:GRM251-500G)
  9. 99.99%二甲基亚砜(DMSO)(Merck,目录号:1.07046.0521)
  10. 抗真菌剂:Dithane R M-45 75%WP(Dow AgroSciences)
  11. 磷酸二氢钠脱水物(NaH 2 PO 4•2H 2 O)(Merck,CAS号:200-664-3) >
  12. 无水磷酸氢二钠(Na 2 HPO 4)(Merck,CAS号:231-448-7)
  13. 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑鎓染料(MTT)(Sisco Research Laboratories,目录号:33611)
  14. 70%酒精(分析Z试剂,弘扬化学,目录号:15005-51)
  15. 去离子水
  16. 50 mM磷酸钠缓冲液(pH 7)(见食谱)
  17. 1个Hoagland的解决方案(见食谱)
    1. 磷酸二氢钾(KH 2 PO 4)(Sisco Research Laboratories,CAS号码:7778-77-0)
    2. 硝酸钾(KNO 3)(Merck,CAS号:231-818-8)
    3. 硝酸钙四水合物(Ca(NO 3)2•4H 2 O)(Merck,CAS号:233-332-1) />
    4. 硫酸镁七水合物(MgSO 4•7H 2 O)(Sisco Research Laboratories,目录号:85611)
    5. 硫酸锰一水合物(MnSO 4•2H 2 O)(Sisco Research Laboratories,目录号:12386)
    6. 七水硫酸锌(ZnSO 4•7H 2 O)(Sisco Research Laboratories,目录号:75738)
    7. 硫酸铜五水合物(CuSO 4•5H 2 O)(Merck,CAS号:231-847-6)
    8. 钼酸铵一水化物((NH4)6Mo7O24•2H2 / > O)(Sisco Research Laboratories,目录号:69429)
    9. 硼酸(H 3 BO 3)(Sisco Research Laboratories,MDL编号:22311)
    10. 氯化铁(FeCl 3)(Thermo Fisher Scientific,目录号:MFCD00011005)
  18. 应激诱导剂(见食谱)

设备

  1. 量筒(Tarsons,产品目录号:345040,345070)
  2. 称重天平(Wensar,型号:PGB 610)
  3. pH计(环球电子,型号:DPH 500)
  4. UV-VIS分光光度计(Techcomp,型号:UH5300)
  5. 离心机(Eppendorf,型号:5810R,目录号:3334)

程序

  1. 用0.2%dithane-M45对水稻种子进行消毒,用去离子水彻底洗涤至少10-15次,直到没有dithane-M45的刺激性气味。

  2. 在去离子水中放置24小时以吸水。

  3. 在30°C的黑暗中种子发芽72小时
  4. 在营养液(1倍Hoagland's溶液)中,在30°C光照16小时,8小时黑暗的光照条件下,在培养管中培育幼苗6天。
  5. 添加5毫升不同的压力诱导剂(见食谱)直接到含有幼苗的培养管。
    加入5毫升去离子水的对照组,搅拌治疗偶尔充气通过冒泡。

  6. 保持控制和处理组16小时
  7. 从生长溶液中取出幼苗,用棉纸擦干。
  8. 称重〜10毫克新鲜根组织,并转移到一个新的2毫升微量离心管。
  9. 加入1.5毫升的MTT溶液(0.25毫克/升,见食谱),并在黑暗中保持4小时。
  10. 弃去MTT溶液,将根样品放入干净的Petriplates中,不要进一步洗涤,用无菌手术刀将根切成1-2mm(确保从形成的甲le中浸出)。向其中加入0.5ml KOH(2N),并将切割的碎块与KOH溶液一起转移到2ml微量离心管中。
  11. 每管加入0.5 ml 99.99%DMSO溶液,使最终体积为1 ml。解决方案的颜色如图1所示。
  12. 在室温下将管子短暂离心5分钟,使根部沉降下来。
  13. 将清澈的上清液转移到新鲜的试管中。
  14. 立即在570纳米的分光光度读数的上清。由于形成的甲photosens的光敏性,始终将样品保持在黑暗中。 (见注1和注2)
  15. O.D.获得的值是10毫克组织,计算O.D.值为1毫克。从O.D.值使用下面的公式来确定细胞死亡的百分比,



    因此,细胞活力/新鲜组织的百分比= 100 - 细胞死亡的百分比

    表1和图2显示了细胞死亡的百分比

    图1.MTT测定的不同阶段的图示:用200mM甘露醇处理的M-空白,共对照,M-处理,用200mM氯化钠处理的Na处理,用200μgnZ处理的mg / L nZVI,用100mM氯化镉处理Cd。 A.在生长室中生长的幼苗; B.用胁迫诱导剂处理16小时后的幼苗; C. MTT测定前的根; D. MTT处理4小时后的根。 Co(i),Cd(i),M,Na,nZ,Co(ii),Cd(ii)根切成1-2mm长。 E.在DMSO中溶解后的甲Color颜色强度。

    表1. O.D.值和细胞死亡百分比



    图2.根据570nm处的OD值确定根组织的细胞死亡百分比。对于100mM CdCl 2 ,细胞死亡百分比最大接着200mg Nzvi> 200mM NaCl> 200毫克甘露醇。

笔记


  1. 添加MTT后避免直射光线,因为它是光敏的
  2. 在DMSO中溶解甲an后立即记录吸光度,因为颜色强度可能会改变,并给出错误的读数。

食谱

  1. 0.2%dithane溶液

    溶解100毫升高压灭菌去离子水200毫克的二甲烷
  2. 50mM磷酸钠缓冲液(pH7)
    1. 准备50mM磷酸二氢钠二水合物(NaH 2 PO 4•2H 2 O)(Merck),通过将0.78g磷酸二氢钠溶于100毫升去离子水
    2. 通过将0.7g磷酸氢二钠溶于100ml去离子水中来制备50mM无水磷酸氢二钠(Na 2 HPO 4)(Merck) >
    3. 55毫升50mM磷酸二氢钠二水合物和45毫升50毫米无水磷酸二氢钠混合制成50毫摩尔磷酸钠缓冲液并调节pH至7
  3. MTT染料
    通过将MTT溶解在50mM磷酸盐缓冲液(pH7)中制备MTT(10mg / ml)的储备溶液。
  4. 2N氢氧化钾溶液
    将11.2克氢氧化钾溶于100毫升去离子水中
  5. 1X Hoagland的解决方案
    宏量营养素:
    1mM磷酸二氢钾(KH 2 PO 4)
    5mM硝酸钾(KNO 3)
    5mM硝酸钙Ca(NO 3)2←2 微量营养素:
    11.8μM硫酸锰一水合物(MnSO 4•2H 2 O)
    0.70μM七水硫酸锌(ZnSO 4•7H 2 O)
    0.32μM硫酸铜五水合物(CuSO 4•5H 2 O)
    0.16μM钼酸铵一水合物((NH4)6Mo7O24•2H2 < / sub> O)
    46.3μM硼酸(H 3 BO 3)
    5μM氯化铁(FeCl 3)
    用蒸压去离子水补足1升
    调整pH值到5.8
  6. 压力诱导剂
    200毫摩尔甘露醇
    200毫克/升nZVI
    100mM CdCl 2•/ 2 200 mM NaCl
    溶于去离子水

致谢

作者希望感谢仪器设备的加尔各答大学第七阶段高级研究中心植物学系。 SM和TG希望承认DBT,GOI和CSIR的财务援助。作者声明没有任何利益冲突。

参考

  1. Carmichael,J.,DeGraff,W.G.,Gazdar,A.F。,Minna,J.D。和Mitchell,J.B。(1987)。 评估基于四氮唑的半自动比色测定:化学敏感性测试的评估 < em> Cancer Res 47(4):936-942。
  2. Kaundal,A.,Rojas,C.M。和Mysore,K.S。(2012)。 测量植物中的NADPH氧化酶活性 Bio Protoc 2(20) :e278。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Majumdar, S., Guha, T. and Kundu, R. (2017). MTT Assay for Cytotoxicity Assessment in Oryza sativa Root Tissue. Bio-protocol Bio101: e2620. DOI: 10.21769/BioProtoc.2620;
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