MTT remarks

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Information about MTT remarks

Published on February 26, 2014

Author: teymourim901


MTT remarks: MTT remarks Presented by:teymouri manouchehr Body surface area: Body surface area The most commonly used formula now is that of Mosteller , published in 1987 in  The New England Journal of Medicine . "normal" BSA is generally taken to be 1.7 m2 Average BSA for adult men: 1.9 m2 Average BSA for adult women: 1.6 m2 Average BSA for children (9 years): 1.07 m2 Average BSA for children (10 years): 1.14 m2 Average BSA for children (12-13 years): 1.33 m2 What does this have to do with mTT Assay?: What does this have to do with mTT Assay? The more drug dose TO the less drug dose Experiment: Experiment A test under controlled conditions that is made to demonstrate a known truth, examine the validity of a hypothesis, or determine the efficacy of something previously untried. Control conditions Simulation Cell Drug MTT Principle: MTT Principle NAD(P)H-dependent cellular oxidoreductase enzyme WST or water-soluble tetrazolium salt: WST or water-soluble tetrazolium salt Phenazine methosulfate (PMS) MTT process: MTT process Cell seeding Cells are ready for treatment Treatment includes serial drug dilution of 2-fold Adding MTT reagent stock (MTT in RPMI Without phenol red Solubilization DMSO SDS in Diluted HCl Acidified ethanol Variables: Variables Cell=10 Drug=10 Variables: Variables Cell=6 These cells needs 2 Hits Drug=12 Metabolized Drug=8 Total drug=12+8 Variables: Variables Cell=3 These cells needs 3 Hits Drug=12 Metabolized Drug=8 Total drug=12+8 Variables: Variables The number of cells in cell seeding The mole or the number of every single drug v.s cells The treatment period Pharmacokinetic concepts: The drug exits in body forever. Every substance at a given concentration is toxic. There are some cells that are resistant to even very high dosage of the drug . Calcusyn: Calcusyn D=dose of drug Fa =The fraction affected by the dose Fu=fraction unaffected =1-fa Effect= fa / fu Effect: Effect Cytotoxicity effect= (the positive control absorbance –Absorbance of the wells treated with every drug dilution )/(The positive control absorbance-the negative control absorbance) The positive control absorbance = the absorbance of the wells that are seeded by cells but received no treatment, in other words, it means 0% toxicity The negative control absorbance= the absorbance of the wells that are not seeded, in other words, it means 100% toxicity Survivability Effect= 1- toxicity effect MTT assay at measuring toxicity: Necrosis =pseudo positive The viable cells metabolically inactive= pseudo negative How to enter data for a single drug: How to enter data for a single drug How to enter data for a single drug: How to enter data for a single drug How to enter data for a drug combination: How to enter data for a drug combination Statistical consideration: Statistical consideration Median-effect equation X Y Yint Drug reduced index=DRI interpretation: interpretation X-intercept= log( Dm ) Dm = IC50 or ED50 Interpretation: Interpretation m value m=1 means Hyperbolic m>1 Sigmoidal m<1 Negative sigmoidal shape Combination index or CI CI=1 means additive CI>1 antagonism CI<1 Synergism Isobologram PowerPoint Presentation: Reutine Invention PowerPoint Presentation: Thanks a lot

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