Mtt lecture

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  • Viable cells that active mitochondria reductase which produce the purple formazan crystals\n
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  • Mtt lecture

    1. 1. MTT Assay
    2. 2. MTT AssayPurpose: To understand how theMTT asay works, what it measures, and why it is important.
    3. 3. How it works:The MTT assay is a colorimetric assay systemand is a quantitative method used to determinecell growth and viability.
    4. 4. Why is the MTT assay important: measuring cellular proliferation (cell growth) in response to external factors
    5. 5. Applications:•Drug sensitivity•Cytotoxicity•Response to growth factors•Cell activation
    6. 6. Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, atetrazole) is reduced to purpleformazan in the mitochondria ofliving cells. (Formazan dyes areartificial chromogenic substrates fordehydrogenases and reductases).
    7. 7. In mitochondria :
    8. 8. - + - - + + ++ + + ++ + ++ + + + - + - + + - + - + + + + + + ++ + + + + + + - + + + + + + + + -+ - + + + + + + + + + + + + + + - + -+ + + + - - + ++ + ++ + + ++ - + + + +
    9. 9. Viable Cell -+ + + + + + ++ - + + + + + -+ - + + + - + + + + + + + +
    10. 10. Non-viable Cell - + - + + - + - + + + + + + ++ + + + + + + - + + + + + + + + -+ - + + + + + + + + + + + + + + - + -+ + + + - - + ++ + ++ + + ++ - + + + +
    11. 11. DMSO+-++ ++ +
    12. 12. Actinomycin D is a substance that binds to DNAand prevents transcription.In this assay it serves as a negative control byinhibiting WEHI cell replication during theincubation time and ensures maximum assaysensitivity.
    13. 13. Count Adjust count cells to 2x105cells/ mL WEHI cells, Mɸ cells, use use RPMI- RPMI-1640 WEHI WEHI+ActD
    14. 14. Wednesday Before Lecture MTT10 15 10µµl µl l
    15. 15. DMSOToday 100 µl
    16. 16. Today Read Incubate for absorbance 5’ in the at 570nm dark

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