Вы находитесь на странице: 1из 2

11.02.

2013, Monday Lab Module: Plant Cell Technology(BEL 712)

Aim: Determination of Cell Viability of plant cells exposed to different hydrodynamic stress. Theory: Hydrodynamic stress has mostly negative effects on cells, these effects can be designated as damage. Various symptoms of cell damage are : alteration of morphology, release of intracellular compounds, alteration of metabolism and productivity, and loss of viability. In this experiment, we will check the viability of Azadirachta indica cell suspension, exposed to different rotational speeds, and hence different hydrodynamic stresses. Triphenyl tetrazolium chloride, TTC, or simply tetrazolium chloride (2,3,5-triphenyl-2H-tetrazolium chloride) is a redox indicator commonly used in biochemical experiments especially to indicate cellular respiration and hence viability. It is a white crystalline powder, soluble in water, ethanol and acetone but insoluble in ether.

TTC assay
In the TTC assay (also known as TTC test or tetrazolium test), TTC is used to differentiate between metabolically active and inactive tissues. The white compound is enzymatically reduced to red TPF (1,3,5-triphenylformazan) in living tissues due to the activity of various dehydrogenases (enzymes important in oxidation of organic compounds and thus cellular metabolism), while it remains as white TTC in areas of necrosis since these enzymes have been either denatured or degraded. For this reason, TTC has been employed in many tissue culture labs to check viability of cells. Note: TTC is somewhat heat and light unstable, so avoid these environments as much as possible.

1 BEL 712

11.02.2013, Monday Reagents: A. TTC vital stain preparation: a. 0.05 M phosphate buffer.

Dissolve 3.403 g KH2PO4 in about 400 ml H2O. Adjust the pH to 5.80 with KOH solution. This will take about 2.4 ml of I N KOH. Bring volume to 500 ml. Re-adjust pH if necessary. b. TTC stain.

Dissolve 1 g of 2,3,5-triphenyltetrazolium chloride in 100 ml of the above buffer. The pH will measure about 4.9. Filter-sterilize and refrigerate in a dark bottle. The stock may be used for several months. B. 95% Ethanol

Protocol: 1. Place 50-100mg of Fresh Weight Plant Tissue in a test tube. 2. Add 3ml of TTC vital stain 3. Infiltrate under vacuum for 15 mins 4. Incubate at 28oC overnight 5. Add 7ml 95% Ethanol and extract the colored complex by boiling in a water bath for 5 mins 6. Mix thoroughly, and centrifuge at 1000g to precipitate the cell debris 7. Measure the absorbance of the supernatant at 490nm 8. Express viability as ABSORBANCE490nm/g-FW.

2 BEL 712

Вам также может понравиться