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Category 'Protocols'
Status: Unanswered
Views: 2987

Quenching Fluorescence using Trypan Blue

Graham Pockley (Author)

Wednesday, February 12, 2014 8:58 AM

We need to confirm or exclude internalisation of a carboxyfluorescein conjugated molecule into tumour cells and are having problems achieving this using the published protocols for the use of Trypan Blue.

Any help / suggestions would be appreciated.

Graham

Michael Daws

Wednesday, February 12, 2014 9:22 AM

I have never managed to get quenching with trypan blue to work very effectively. An alternative is to use a secondary reagent to counterstain molecules that have not been internalised - in your case you could use an anti-fluorescein secondary reagent. Everything that is internal will just have fluorescein staining, whilst everything that is on the surface will also stain with the secondary. This works best for imaging flow cytometry, where you get a picture of each cell, but can also work with flow cytometry by looking at the ratio of staining in the two channels.