Evaluating integrin activation with time-resolved flow cytometry

Jesus Sambrano; Alexandre Chigaev; Kapil S. Nichani; Yelena Smagley; Larry A. Sklar; Jessica P. Houston

doi:10.1117/1.JBO.23.7.075004

10 July 2018 Evaluating integrin activation with time-resolved flow cytometry

Jesus Sambrano, Alexandre Chigaev, Kapil S. Nichani, Yelena Smagley, Larry A. Sklar, Jessica P. Houston

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Journal of Biomedical Optics, Vol. 23, Issue 7, 075004 (July 2018). https://doi.org/10.1117/1.JBO.23.7.075004

Abstract

Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4β1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Jesus Sambrano, Alexandre Chigaev, Kapil S. Nichani, Yelena Smagley, Larry A. Sklar, and Jessica P. Houston "Evaluating integrin activation with time-resolved flow cytometry," Journal of Biomedical Optics 23(7), 075004 (10 July 2018). https://doi.org/10.1117/1.JBO.23.7.075004

Received: 26 March 2018; Accepted: 13 June 2018; Published: 10 July 2018

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