Imaging nanoBiT

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  • Last Post 30 November 2017
ebaxter67 posted this 06 October 2017

So how best to see nanoBiT interactions under the microscope. I can get plasmids into cells and see strong bioluminescence signals on plate reader but nothing on the microscope. Should it be visible tothe naked eye once the lights are off?

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alandrem posted this 06 October 2017

Hello @ebaxter67

We have successfully imaged the NanoBiT interactions, but have done so using a microscope specifically developed for bioluminescent imaging. Although the NanoBiT signal is bright, compared to fluorescent signals it is still often not bright enough to see on a typical fluorescent microscope set up because there are usually quite a few filters in the light path that block  a lot of the signal. What type of scope are you using?

 

ebaxter67 posted this 10 October 2017

Hi,

 

Thanks very much for the comment - I was using a Nikon TiE and a CCD camera so perhaps there's not enough light coming through like you suggest. Our next move is to try the cells with a Nikon A1R in a confocal set-up - I imagine the light gathering will be better? Have you been using the Olympus LV 200 I heard about through oour local Promega rep?

 

alandrem posted this 27 October 2017

Hi! So sorry I missed this. Yes, we have been using the Olympus LV200 in our work. How did your next try work out?

 

ebaxter67 posted this 30 November 2017

I guess my next question is how much should I be getting from about 20000 cells in a well of a 96 well plate. I can get about 10,000 counts using a 10 second time period on a Berthold plate reader when I cross link my dimer partners together using F(ab')2 fragments. Do you have any feel for whether this is good, bad or indifferent?

 

I hope my stable transfectants will make a difference in the amount of each partner expressed and hence how bright the cells are with furimazine.

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