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. -
+
-
-
+
+
+
+
+
+
+
+ +
+
+ +
+
+
- +
- +
+ -
+
-
+ +
+
+ +
+
+
+
+ +
+
+
+
+ -
+
+
+
+
+
+
+
+
-+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
- +
-+
+
+
+
-
-
+ +
+
+
+
+
+
+
+
+
-
+
+
+
+

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