Mtt lecture
- 1. MTT Assay
- 2. MTT Assay Purpose: To understand how the MTT asay works, what it measures, and why it is important.
- 3. How it works: The MTT assay is a colorimetric assay system and is a quantitative method used to determine cell growth and viability.
- 4. Why is the MTT assay important: measuring cellular proliferation (cell growth) in response to external factors
- 5. Applications: •Drug sensitivity •Cytotoxicity •Response to growth factors •Cell activation
- 6. Yellow MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells. (Formazan dyes are artificial chromogenic substrates for dehydrogenases and reductases).
- 8. - + - - + + + + + + + + + + + + + + - + - + + - + - + + + + + + + + + + + + + + - + + + + + + + + -+ - + + + + + + + + + + + + + + - + -+ + + + - - + + + + + + + + + + - + + + +
- 9. Viable Cell -+ + + + + + + + - + + + + + - + - + + + - + + + + + + + +
- 10. Non-viable Cell - + - + + - + - + + + + + + + + + + + + + + - + + + + + + + + -+ - + + + + + + + + + + + + + + - + -+ + + + - - + + + + + + + + + + - + + + +
- 11. DMSO +-+ + + + +
- 12. Actinomycin D is a substance that binds to DNA and prevents transcription. In this assay it serves as a negative control by inhibiting WEHI cell replication during the incubation time and ensures maximum assay sensitivity.
- 13. Count Adjust count cells to 2x105cells/ mL WEHI cells, Mɸ cells, use use RPMI- RPMI-1640 WEHI WEHI+ActD
- 14. Wednesday Before Lecture MTT 10 15 10µ µl µl l
- 15. DMSO Today 100 µl
- 16. Today Read Incubate for absorbance 5’ in the at 570nm dark