Dear Alif,

Take the average samples replicate values.

Subtract the blank value of this average.

Divide (sample1/control)×100%

You provide more details, would be helper to understand a value bigger than 100% if you expect to get less.

Hi Mohamed Khashan , My experimental design is as follow :

I induced oxidative stress on HEK293 cells and treat them with my sample afterwards. I am expecting my sample to be able to protect my cells from oxidative stress death hence , I would expect it to be more.

Is there any other way to express this MTT Assay result other than this way? I was thinking on reduction/increase in cell viability as well but I am not too sure.

Thanks for your suggestion!

Dear Alif Ismail ,

I'm not sure, if MMT is the right assay for your set up. Since MTT in reduced in the mitochondria to formazan I would have doubts that your oxidative stress is not interfering with the reaction or mitochondrial activity. You might find this article helpful:

https://www.mdpi.com/1422-0067/22/23/12827

best wishes

Soenke

Hi Sönke Weinert . Thank you so much for the input. I've read the article you sent and it's informative to know the limitation of this MTT assay.

To clarify my experimental set up; I am challenging my HEK293 cells with hydrogen peroxide and I want to investigate potential protective effect of my stingless bee honey samples. Apart from that, I am also trying to see if my samples could promote recovery of cell upon inflammation induced by oxidative stress from hydrogen peroxide (for this purpose, i incubated my cells with the hydrogen peroxide first before removing them and replaced with media+honey samples).

I do expect that there will be reduced mitochondrial activity upon oxidative damage by the hydrogen peroxide in the cells. However, I am interested to see if there's any different between cell viability between the one without my honey samples and the one with my honey sample. Therefore, i expect that those cells with reduced mitochondrial activity (dying cells) upon induction of oxidative stress will be more in the untreated cells group.

Hence, I think the effect of oxidative stress to the mitochondrial activity is not much of a concern for me.

However, may I ask some suggestions? Based on my experimental set up and aim, do you think my approach is right to address them? If not, could you suggest a proper experimental setup, or another method?

Thank you so much for your time to reply and giving input! I really appreciate them.

Dear Alif Ismail ,

I'm not really in the field of viability measurements. Of cause I have done some during my professional career, but I would not consider me as an expert in the field.

Depending on your lab equipment, you might want to try an ATP or CFSE assay or a live dead staining. You can find an overview here:

https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/imaging-analysis-and-live-cell-imaging/cell-viability-and-proliferation?gclid=CjwKCAjwkeqkBhAnEiwA5U-uM5KmM35BrDFyPp00wxnMsN4S9pO3MN8AZeKsr5Ap6JW9FD9Gmoef4xoCriwQAvD_BwE

best wishes

Soenke

Hi Alif Ismail

I would recommend you to use alamarBlue dye to measure cell viability as it is completely non-toxic and does not interfere with mitochondria as MTT.

Please find the attached links for in-depth details on the same.

1. Article Comparison of Alamar Blue and MTT assays for high throughput screening

2. https://www.bio-rad-antibodies.com/alamarblue-advantages-best-in-vitro-assay.html?JSESSIONID_STERLING=556EE1A64C7E2FCD9D6C727C42BF29BB.ecommerce2&evCntryLang=IN-en&cntry=IN&thirdPartyCookieEnabled=true

Thanks,