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MTT Assay for Cytotoxicity Assessment in Oryza sativa Root Tissue
Published: Nov 20, 2017 DOI: 10.21769/BioProtoc.2620 Views: 8858
Edited by: Arsalan Daudi Reviewed by: Jian Chen
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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.
Cytotoxicity
MTT assay
Plant tissue
Rice seedling
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
- Culture tubes (Borosil, catalog number: 9910010 )
- Tissue paper (Kim wipes) (Tarsons, catalog number: 370080 )
- 2 ml microcentrifuge tube (gem. gov. in, Genaxy, catalog number: GEN-MT-200-C )
- Aluminium foil FreshwrappR (Hindalco)
- Petri plates (Borosil, catalog number: 3160065 )
- Rice Oryza sativa cv. IR64 (6 days old) (Rice Research Station Government of West Bengal, Chinsurah R.S., Hooghly, India)
- Stress inducing agents:
- Nanoscale zero-valent iron nanoparticles (nZVI) (gifted by Prof. A. Mukherjee, Vellore Institute of Technology, Tamil Nadu, India)
- Cadmium chloride monohydrate (CdCl2·H2O) (Merck, catalog number: 61813101001730 )
- Sodium chloride (NaCl) (HiMedia Laboratories, catalog number: MB023-500G )
- Mannitol(C6H14O6) (Sisco Research Laboratories, catalog number: 134889 )
- Nanoscale zero-valent iron nanoparticles (nZVI) (gifted by Prof. A. Mukherjee, Vellore Institute of Technology, Tamil Nadu, India)
- Potassium hydroxide (KOH) (HiMedia Laboratories, catalog number: GRM251-500G )
- 99.99% dimethyl sulfoxide (DMSO) (Merck, catalog number: 1.07046.0521 )
- Antifungal agent: DithaneR M-45 75% WP (Dow AgroSciences)
- Sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O) (Merck, CAS number: 200-664-3)
- Di-sodium hydrogen phosphate anhydrous (Na2HPO4) (Merck, CAS number: 231-448-7)
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) (Sisco Research Laboratories, catalog number: 33611 )
- 70% alcohol (Analytical Z Reagent, Hong Young Chemicals, catalog number: 15005-51 )
- Deionised water
- 50 mM sodium phosphate buffer (pH 7) (see Recipes)
- 1x Hoagland’s solution (see Recipes)
- Potassium dihydrogen phosphate (KH2PO4) (Sisco Research Laboratories, CAS number: 7778-77-0)
- Potassium nitrate (KNO3) (Merck, CAS number: 231-818-8)
- Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (Merck, CAS number: 233-332-1)
- Magnesium sulphate heptahydrate (MgSO4·7H2O) (Sisco Research Laboratories, catalog number: 85611 )
- Manganese sulphate monohydrate (MnSO4·H2O) (Sisco Research Laboratories, catalog number: 12386 )
- Zinc suphate heptahydrate (ZnSO4·7H2O) (Sisco Research Laboratories, catalog number: 75738 )
- Copper sulphate pentahydrate (CuSO4·5H2O) (Merck, CAS number: 231-847-6)
- Ammonium molybdate monohydrate ((NH4)6Mo7O24·H2O) (Sisco Research Laboratories, catalog number: 69429 )
- Boric acid (H3BO3) (Sisco Research Laboratories, MDL number: 22311 )
- Ferric chloride (FeCl3) (Thermo Fisher Scientific, catalog number: MFCD00011005 )
- Potassium dihydrogen phosphate (KH2PO4) (Sisco Research Laboratories, CAS number: 7778-77-0)
- Stress inducing agents(see Recipes)
Equipment
- Measuring cylinder (Tarsons, catalog numbers: 345040 , 345070 )
- Weighing balance (Wensar, model: PGB 610 )
- pH meter (Global Electronics, model: DPH 500 )
- UV-VIS spectrophotometer (Techcomp, model: UH5300 )
- Centrifuge (Eppendorf, model: 5810R , catalog number: 3334)
Procedure
Category
Plant Science > Plant cell biology > Tissue analysis
Cell Biology > Cell viability > Cell death
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