#cell viability

Plant tissue culture 2

Dead protoplasts are stained red by:

Dead protoplasts are stained red by propidium iodide PI nucleic acid intercalating fluorescent dye that distinguishes membrane compromised cells. PI is cationic molecule unable to cross intact lipid bilayer of viable protoplasts due to charge and size exclusion, so viable population excludes dye remaining unstained. In dead cells membrane integrity lost through enzymatic damage or physical stress, PI diffuses entering cytoplasm nucleus where it intercalates between double stranded DNA and RNA base pairs with strong affinity, enhancing red fluorescence emission at 617 nm when excited green ligh

Ref: Jones 1987 PI dead staining; Molecular Probes Handbook viability dyes.

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Plant tissue culture 2

Viable protoplasts fluoresce green when stained with:

Viable protoplasts fluoresce bright green when stained with fluorescein diacetate FDA, vital fluorescent dye used routinely for viability assessment before fusion or transformation to ensure batch quality above 80 percent. FDA itself is non fluorescent lipophilic molecule that diffuses across intact plasma membrane permeable only to living cells with intact barrier. Inside cytoplasm active non specific esterases present exclusively in living cells hydrolyze ester bonds releasing fluorescein highly fluorescent polar molecule that accumulates inside membrane intact vesicle because its negative c

Ref: Widholm 1972 FDA viability; Larkin 1976 fluorescein diacetate Plant Physiol.

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Animal Tissue Culture 3

The purple color in MTT assay is due to formation of:

Purple color appearing in wells after MTT incubation arises from formation of formazan product 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan. Enzymatic reduction transfers electrons from succinate via succinate dehydrogenase, NADH and NADPH to tetrazolium ring breaking positively charged quaternary nitrogen and producing neutral formazan with extensive conjugated double bond system delocalizing electrons, absorbing green-yellow wavelengths around 570 nm and appearing purple. Crystals observable microscopically as dark needle-like precipitates clustered around nucleus and in cytoplasm befor

Ref: Berridge et al Arch Biochem Biophys 2005 MTT formazan formation mechanism; Abcam MTT assay protocol formazan purple product succinate dehydrogenase.

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Animal Tissue Culture 3

MTT assay measures:

MTT assay developed by Tim Mosmann in 1983 measures cell viability and proliferation based on reduction capacity of living cells. Principle uses yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide which enters cells via endocytosis and is reduced by mitochondrial succinate dehydrogenase of complex II and cytosolic NAD(P)H-dependent oxidoreductases cleaving tetrazolium ring with electrons from NADH and NADPH producing insoluble purple formazan crystals accumulating intracellularly in mitochondria and lipid droplets. Metabolically incompetent dead cells fail to r

Ref: Mosmann T J Immunol Methods 1983 MTT viability assay; ATCC MTT Cell Proliferation Assay mitochondrial dehydrogenase reduction protocol.

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Animal Tissue Culture 3

Cell viability during log phase is approximately:

Exponential log phase represents period of maximal health for mammalian cultures. Under optimal conditions of adequate nutrients, pH maintained at 7.2-7.4 by CO2-bicarbonate buffering, temperature 37°C, and absence of waste accumulation, trypan blue exclusion tests reveal membrane intact viable cells at 90-100% because apoptotic signaling minimal, caspase-3 inactive, mitochondrial membrane potential high as indicated by JC-1 aggregation. Mitochondrial succinate dehydrogenase activity robust leading to efficient reduction of tetrazolium salts in MTT assay. Cells display high cloning efficiency

Ref: Freshney Ch.18 Viability during log 90-100%; ATCC Cell Viability trypan blue exclusion exponential phase near maximal.

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