High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry

Nikoletta Szalóki, Quang Minh Doan-Xuan, J. Szöllősi, Katalin Tóth, G. Vámosi, Z. Bacsó

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.

Original languageEnglish
Pages (from-to)818-829
Number of pages12
JournalCytometry Part A
Volume83
Issue number9
DOIs
Publication statusPublished - 2013

Fingerprint

Laser Scanning Cytometry
Fluorescence Resonance Energy Transfer
Proteins
Flow Cytometry
Image Cytometry
Photobleaching
HeLa Cells
Confocal Microscopy

Keywords

  • Fluorescence resonance energy transfer
  • FRET
  • High throughput
  • Laser scanning cytometry
  • Protein-protein interactions

ASJC Scopus subject areas

  • Cell Biology
  • Histology
  • Pathology and Forensic Medicine

Cite this

High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry. / Szalóki, Nikoletta; Doan-Xuan, Quang Minh; Szöllősi, J.; Tóth, Katalin; Vámosi, G.; Bacsó, Z.

In: Cytometry Part A, Vol. 83, No. 9, 2013, p. 818-829.

Research output: Contribution to journalArticle

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