The MTT assay is a quantitative method to measure cell growth and viability. It works by using the yellow tetrazolium dye MTT, which is reduced to purple formazan in the mitochondria of living cells. The amount of formazan produced is proportional to the number of viable cells and can be measured spectrophotometrically. The MTT assay is important for measuring cellular proliferation in response to external factors and has applications in testing drug sensitivity, cytotoxicity, and cell responses to growth factors.

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).

7. In mitochondria :

8. -
+
-
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+
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+ +
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+ +
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- +
- +
+ -
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+ +
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+ +
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+ -
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-
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- +
-+
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+ +
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+

9. Viable Cell
-+
+
+
+
+
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+ -
+
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-
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10. Non-viable Cell
- +
- +
+ -
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+ +
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+ +
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+ -
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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