Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry

Background: Flow cytometry may be used to isolate large amounts of living, fluorescently labeled cells. Certain fluorescent labels, like enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), allow the assessment of direct protein–protein interaction in situ, by fluorescence resonance energy transfer (FRET). However, current flow cytometric methods either require elaborate technical adaptations or, using a single laser protocol, are hampered by background signal. We optimized a single 408‐nm laser protocol to detect FRET between ECFP/EYFP‐tagged proteins. Methods: Cell lines stably expressing ECFP and/or EYFP or an EYFP‐ECFP fusion protein were used to design the settings for the flow cytometer to detect FRET‐positive cells using a single 408‐nm laser. Using these settings, interactions between the subunits of the transcription factor NF‐Y were studied. Results: Flow cytometric analysis of the cells expressing an EYFP‐ECFP fusion protein yielded a discrete FRET‐positive population. Using the same settings, in cells expressing NF‐YB‐CFP and NF‐YC‐YFP fusion proteins, FRET could also be detected. These cells were sorted and FRET was confirmed by confocal microscopy. Conclusion: FRET‐positive cells, expressing ECFP‐ and EYFP‐tagged proteins, can be detected using single 408‐nm laser excitation, with low background signal. This allows high‐throughput analysis and isolation of viable FRET‐positive and ‐negative cells for subsequent biological experiments. © 2006 International Society for Analytical Cytology