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MTT Assay for Cytotoxicity Assessment in Oryza sativa Root Tissue   

Abstract

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 Freshwrapp^R (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: Dithane^R 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

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2. Kaundal, A., Rojas, C. M. and Mysore, K. S. (2012). Measurement of NADPH oxidase activity in plants. Bio Protoc 2(20): e278.