Computational protocol: Unifying Concept of Serotonin Transporter-associated Currents*

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Protocol publication

[…] Resonance energy transfer (FRET) measurements in HEK293 cells expressing fluorescently tagged hSERT were performed as described previously (), using a Zeiss Axiovert 200 microscope. In brief, images were taken using a CCD camera (Coolsnap fx; Roper Scientific), a 63× NA 1.4 objective, and LUDL filter wheels allowing a rapid excitation and emission filter exchange. Background fluorescence was subtracted from all images. We analyzed the images pixel by pixel using National Institutes of Health ImageJ 1.43b with the plug-in PixFRET (). Spectral bleed-through parameters for the donor bleed-through (DBT) and the acceptor (ABT) were determined, and NFRET was calculated according to . For statistical comparison the mean NFRET was measured at the plasma membrane using the computed NFRET image. […]

Pipeline specifications

Software tools ImageJ, PixFRET
Applications Microscopic phenotype analysis, FRET
Organisms Homo sapiens
Chemicals Potassium, Serotonin