PRINCIPLE AND APPLICATIONS OF CELL VIABILITY, GLUCOSE UPTAKE AND CALCIUM UPTAKE ASSAYS

Introduction to Terms, Cell Viability Assay, Glucose Uptake Assay, Calcium Uptake Assay
Presented by
B. KRANTHI KUMAR
Department of Pharmacology

1. 1 PRINCIPLE AND APPLICATIONS OF CELL VIABILITY, GLUCOSE UPTAKE AND CALCIUM UPTAKE ASSAYS A Seminar as a part of curricular requirement for I year M. Pharm I semester Presented by B. KRANTHI KUMAR (Reg . No. 20L81S0108) Under the guidance/Mentorship of Dr. K. SOMASEKHAR REDDY M.Pharm , Ph.D , Associate Professor And Head Dept . of Pharmacology

2. 2 Contents TERMS CELL VIALBILITY ASSAY Tetrazolium reduction assay MTT tetrazolium assay concept Real time viability assay ATP assay Applications GLUCOSE UPTAKE ASSAY Principle Procedure Applications CALCIUM UPTAKE ASSAY Principle Procedure Applications References

3. 3 TERMS An assay is a process of analyzing a substance to determine its composition or quality. Cell viability ,defined as the number of healthy cells in a sample, determines the amount of cells that are living or dead, based on a total cell sample. CELL VIALBILITY ASSAY This is a homogenous method used to estimate (or) determine the number of viable cells in culture (or) multi-well plate. The mainly used CVA’s - Tetrazolium Reduction Assay (TRA), Real Time Viability Assay(RVA), ATP Assay(ATPA).

4. 4 Tetrazolium Reduction Assay For the detection of viable cells, a variety of tetrazolium compounds are used . They are: MTT, MTS, XTT and WST-1. These compounds fall into two basic categories: MTT which is positively charged and readily penetrates viable eukaryotic cells Those such as MTS,XTT, and WST-1 which are negatively charged and do not readily penetrate cells.

5. 5 MTT Tetrazolium Assay Concept The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay. Principle : This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide or MTT) to purple formazan crystals. The viable cells contain NAD(P)H- dependent oxidoreductase enzymes which reduce the MTT to formazan . MTT FORMAZAN OXIDO REDUCTASE

6. 6 Procedure The MTT substrate is prepared in a physiologically biological solution, added to cells in culture medium, usually at concentration of 5mg/ml, and incubated for 1 to 4hrs. The quantity of formazan measured by recording changes in absorbance at 570nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used. In Detail Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570nm when cells die, they lose the ability to convert MTT into formazan .

7. 7 T he formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to solubilize the formazan product, various solubilization methods include using: acidified isopropanal , DMSO, dimethylformamide , SDS, and combinations of detergent and organic solvent. The pH of the solubilization solution can be adjusted to provide maximum absorbance.

8. 8 The amount of signal generated is dependent on several parametres including: The concentration of MTT The length of the incubation period The number of viable cells and their metabolic activity All of these parameteres should be considered when optimizing the assay conditions to generate a sufficient amount of product that can be detected. The conversation of MTT to formazan by cells in culture is time dependent .

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10. 10 Real time viability assay

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12. 12 Applications CVA are used for measuring the results of cell proliferation Used in testing for cytotoxic effects of compounds and for multiplexing to determine cell viable number. In vitro CVS’s with cultured cells are widely used for cytotoxicty cells of chemicals and for drug screening. Currently these assays are also used in oncological researches.

13. 13 Glucose Uptake Assay Principle Glucose uptake activity was analyzed by measuring the rate of uptake of radioactively tagged 2-deoxy glucose in differentiated 3T3 L1 cells. After starvation the cells were treated with insulin and other plant extracts. Then these ligands will bind to the receptors on the surface of the cells. These triggered the translocation of glucose transporters to the cell surface.

14. 14 Then we treat it with radioactive cocktail containing 10 µ M 2-deoxy glucose and 0.25µ Ci of 2 deoxy -D-(3H)-glucose. So the radioactively tagged glucose will enter the cell along with the normal glucose. By measuring this uptake rate by using liquid scintillation counter help to analyze the glucose uptake activity and the effect of plant extract on the glucose uptake activity.

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16. 16 Procedure

17. 17 Applications To study the effect of insulin and insulin like substance on muscle tissue. To study glycogen synthesis To study glucose transport To study glycogen synthase activity. The glucose uptake assay is a plate-based, homogeneous bioluminiscent method for measuring glucose uptake in cells, based on the detection of 2-deoxyglucose-6-phosphate. Glucose uptake experiments are commonly used to measure cellular metabolic activity and glucose transport. Glucose uptake can be studied using radiolabeled glucose itself, or radiolabeledglucose analogs such as 2-deoxy-D-glucose(DOG) or 3-O-methyl-D-glucose.

18. 18 Calcium Uptake Assay Calcium is a ubiquitous second messenger, involved in everything from the contractility of the cardiac and skeletal muscles to blood clotting, bone mineralization and the functioning of the nervous system. Calcium ions contribute to signal transduction by affecting local electrostatic fields and interacting with proteins to alter their conformations. For instance, calcium is important in the transmission of nerve impluse via neurotransmitter release, but also aids in triggering muscle contraction by communicating with regulatory proteins that allow the interaction of actin and myosin. It even acts as a cofactor for certain enzymes.

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20. 20 Principle The Principle of flow cytometric calcium flux measurment is based on changes in fluorescence intensity or emission wavelength of a fluorophore following chelating of calcium ions. This is commonly reported as a plot of the fluorescence intensity against time.

21. 21 Procedure This method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cells anion transporter are particularly active. The addition of probenicide , an inhibitor of anion transporter, is required for retention of this dye in the cell. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of receptor.

22. 22 Applications Calcium detection assays used to diagnose Bone diseases Osteoporosis Kidney disease Hypercalciuria Thyroid disease Hyperparathyroidism Hypertension Alzheimer’s disease

23. 23 References Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.1983;65:55-63. Mashall N J. Goodwin C J. Holt SJ. A critical assessment of the use of microculture tetrazolium assay to measure cell growth and function. Growth regul.1995;5(2):69-84. Sasson S. Oron R. Cerasi E. Enzymatic assy of 2-deoxyglucose 6- phosphate for assessing hexose uptake rates in cultured cells, Anal. Biochem.1993;215:309-311. Saarabia V. Ramlal T. Klip A. Glucose uptake in human and animal muscle cells in culture. Biochem . Cell Biol.1990:68:536-542.

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