Monitoring of insulin granule packaging in live cells using homoFRET-FP detection

N.Y. Yi (1), Q. He (1), T.B. Caligan (1), G.R. Smith (1), L.J. Forsberg (2), J.E. Brenman (2), J. Z. Sexton (1) (1) North Carolina Central University, (2) UNC Chapel Hill School of Medicine 07/2016

Diabetes mellitus is a metabolic disease arising from resistance to insulin or limited insulin secretion. Due to the high prevalence of diabetes, studying modulators of insulin production and storage is highly demanded. In the course of insulin secretion, the hormone is packed into vesicles, leading to a high insulin density therein. This is exploited for measuring granular insulin using the fluorescently labelled hormone. Highly-concentrated fluorophores of the same type are able to undergo resonance energy transfer (homo-FRET). Polarized excitation light results in randomized emission in case energy was transferred to a neighboring fluorophore (homo-FRET-FP).

This application note shows that detecting homo-FRET-FP of mCherry labelled insulin with the PHERAstar® allows for monitoring of cellular insulin-granules in 384 well plate format. Preventing vesicle formation decreases the FP value, representing lower local mCherry-insulin concentrations and accordingly less emission light randomization.

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