Variable result of MTT assay - (Nov/17/2006 )

Hello guys
I am not getting consistent results, though I tried to maintain equal concentration of cell seeding. Please I need some more tips and constructive suggestions. Thanks in ADAVANCE.

I did MTT assay for HT1080 cells twice. The cancer cells were grown on 96 wells plate (5X10,000/ml). After 24 hrs of drug treatment, MTT 5 mg/ml was added and OD was measured at 540.

Dinesh

---

hi, there's a tendency that your MTT wasn't well mixed?
So I normally let the plate shake in the microplate reader for 5seconds before reading.
Also, for fluorescence/luminiscence readings, you'll need to incubate it for 37degrees before taking the readings. (However, yours is a colorimetric so it should be the case)

Are your HT1080 cells of similar passage? If passage is too far off, it may affect your MTT readins too

---

Hi!
We also add a lysant after MTT incubation. Then we shake it for 5 minutes and then we read the plate.
Incubation time varies according to cell line i.e. adherent/non adherent. Also lysant time changes.
Is your cell line adherent? then incubate from 2 to 5 hours and lysate with DMSO or ethanol after removing surnatant.
Does it grow in suspension? incubate 4 hours and lysate with SDS 10%.
At least, this is what we do!

---

Thanks Sharonpek and Panda.
Furthermore, my cell line HT-1080 is one of the fast growing cells. My prof. always prefer on good pipetting practice for equal seeding. If such, then how is the idea of using 48 wells plate. I just guess, using large number of cells initially, will result less variable results within the intra-groups?????? Did you guys ever try such?????????

---

Hi ThapaDinesh,
Do you happen to be using multichannel pippette? That's the best we can go for proper seeding.


I don't use the larger sized plates because you'll have to add more of the reagents which is really a waste

---

Hello Sharonpek
I have been trying with just micropipette (pipette tip having wide aperture).

I appreciate your idea regarding using larger plate.

Dinesh

---

is it possible to maybe measure protein levels in the wells so that MTT readings can be normalised to protein values. Shouldnt this account for any variablity during cell seeding?

---

Wini,
You got the point for normalization, yet we just dont do in routine MTT assay. Withing the set, the intragroups results are compared for equal cell seeding.

Anyway, do you have any detaied information about normalisation???

Thanks for the response and point.

Dinesh

---

Hi Dinesh,

The cell line that you are using is fast growing. What's the cell number per well? How long you incubate your cells before starting compound treatment? What's a compound vehicle? What solubilization reagent you are using? I am asking too many questions but that will help in tracking down the problem.

---

[/quote]

Hi Dinesh,

The cell line that you are using is fast growing. What's the cell number per well? How long you incubate your cells before starting compound treatment? What's a compound vehicle? What solubilization reagent you are using? I am asking too many questions but that will help in tracking down the problem.
[/quote]

Dear Exploresci
Thanks for your interest. Ok I will try to reply your questions one by one.
Cell number??
3X10000 cells/well or 1.5X100000 cells/ml
Incubation before treatment??
18-24 hrs
Vehicle?
DMSO
Solublizing reagen?
DMSO
i incubate 15-20 minutes at 37 for the solubalization of crystals before taking OD in 540nm.

Moreover, I am willing to know the toxicity level of DMSO, the vehicle used for drug treatment.

Thanks again.

---