Luciferin can be used to assess luciferase activity using living cells/tissues. The most frequent use is injection into mice to perform live imaging experiments. The approach should work with cells as well. However, luciferin is quite expensive and large quantities will be needed to get a strong signal. Unless it is critical to maintain cell viability, performing the assay after cell lysis is best. If cell viability is critical, co-expressing luciferin is the most cost-effective option. I am unaware of a commercial kit designed specifically for detecting luciferase activity in vivo or in living cells.

Hi Stephan,

You can just add luciferin to cells and image them. The signal lasts until 30mins and then fades away. I have done this before.

good luck.

Thanks guys!

Roshan, in which quantity/concentration do you add the luciferin? I was intending to have my cells in a 96 well format.

Thanks again :)

Hi Stefan,

You can buy StayBrite™ D-Luciferin sodium salt (MW: 302 g/mol) 1g which can be cheaper compared to smaller quantities and can be used for several assays. Dissolve luciferin in 1x PBS at 15 mg/mL to make 100x stock solution.

For cell culture assays, I usually dilute the luciferin stock in normal cell culture medium, preferably in the absence of phenol-red, to get a final concentration of 0.15 mg/mL, ~500 μM (1x). I used U87MG cells expressing firefly luciferase. I usually seed 10,000 cells per well in a 96 well plate (Transparent bottom, white or black walls).

As Jonathan mentioned, it is better to make the measurement within 30 minutes after the replacement of luciferase containing medium.

Best sensitivity can be obtained if you have IVIS in vivo imaging system. I used CLARIOstar plate reader in well-scanning mode (2 x 2 matrix), top optics, focal height (15 mm), temperature (37° C) and gain (3600).

Thank you, I'll try it in the new year and let you know how it worked out! :)

Hi Stefan

How did this go?