I have XTT powered substance (not kit!) so I have problem finding a proper protocol for applying this in macrophage/lymphocyte cell cultures.
I have looked at many papers and kit protocols where there are a lot of info about assay but few things are still vague to me! I can't find proper concentration of starting XTT solution nor the exact amount of uL applied in well. The absorbance measured after is done at range from 400-500 nm so which one is the best?
Also do I need the activator for XTT that is stated in all kit protocols but there is no mention about the activator in papers.
Dear Nikola,
Pleas find attached my protocol that worked well with Hepa cells. My incubation time with XTT was 4h when my absorbance reading for controls (non-treated cells) was around 0.8, while my blanks were around 0.1 or 0.2 depending on the culture medium.
You will maybe have to adapt your amount of XTT solution (I used 80 ul per well).
I made my reading at 450 nm. It is important that you DO NOT remove your culture medium before adding the XTT solution.
The XTT activator in menadione.
I hope this can help you.
Good luck with your experiments,
Claudia
Dear Nikola,
Pleas find attached my protocol that worked well with Hepa cells. My incubation time with XTT was 4h when my absorbance reading for controls (non-treated cells) was around 0.8, while my blanks were around 0.1 or 0.2 depending on the culture medium.
You will maybe have to adapt your amount of XTT solution (I used 80 ul per well).
I made my reading at 450 nm. It is important that you DO NOT remove your culture medium before adding the XTT solution.
The XTT activator in menadione.
I hope this can help you.
Good luck with your experiments,
Claudia
Be careful with macrophage activation since XTT very good sensor for superoxide, especially without electron cerriers such as PMS or menadione. So, results of XTT test could be strongly affected with ROS production.
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