MTT assay protocol

Viability test. Blue-purple color viable cells.

Yellow-orange color less viable cells.

Higher Absorbance (OD value in!icates an increase in cells viability" lower absorbance
value in!icates a re!uction in cells viability.
#eagents preparation$
MTT %olution$ & 'g('l MTT) in *B%. %olution 'ust be +ilter sterili,e! (-.../' +ilter
a+ter a!!ing MTT. 0or long ter' storage$ 1eep the MTT solution in -.- 2.

MTT %olvent$ option 34$ 5-- ul H2l 4M" 4- 'l triton 4--6" 7-'l isopropanol.
option 3.$ DM%O 4--6. (#oo' Te'p
8otes$
2on+luence o+ the cells 'ust be less than 4--6.
*roce!ure$
4. #e'ove all the culture 'e!iu' +ro' the wells.
.. A!! 7- ul o+ new culture 'e!iu'.
9. A!! 4-- ul *B% to e'pty wells.
- 0ro' this stage wor1 in a !ar1 environ'ent :
5. A!! 4- ul o+ prepare! MTT solution to the treate! wells (a!! at once using a
'ultichannel pipettes : !o not pipette.
0inal volu'e with MTT solution : 4-- ul.
&. 2over in alu'inu' +oil.
;. <ncubate at 9=
o
2 +or . to 5 hours until purple precipitate is visible
(so'eti'es the blue color will be visible only a+ter a!!ing the solvent in stage =.
=. A!! 4-- ul o+ solvent : pipette well.
>. 4& 'inutes in 9=
o
2 incubator.
7. #ea! by ?lisa rea!er.
4-. #ea! absorbance at &;-n' an! also rea! the re+erence at ;&-n'.
44. %ubtract the ;&-n' re+erence rea! +ro' the &;-n' absorbance rea!.
(Value @ &;-n'-;&-n'
) MTT : Thia,olyl blue tetra,oliu' bro'i!e : %ig'a 3'.4.> : store at 5
o
2.
V& Aritten by$ Tatyana an! <tai 9-.7.45
Toren lab