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Published on: December 8, 2015
FRET causing misleading signal from fluorescein excited by the violet laser in flow cytometry
Louis Waeckel1, Hana Khenine2, Anne-Emmanuelle Berger1
1Immunology Lab, University Hospital, Saint-Etienne, France.
Insights
A new fluorescence resonance energy transfer (FRET) interference was identified between Pacific Blue (PB) and FITC fluorochromes, excited by a violet laser. This FRET effect can cause misleading signals in multicolor flow cytometry, especially when labeling closely related markers.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Multiple fluorochromes in immunolabeling can cause spectral interferences.
- Fluorescence Resonance Energy Transfer (FRET) is a known phenomenon that can lead to unexpected signals.
- Previous FRET observations in T cell clonality analysis involved PE-Cy7 and APC-Alexa750.
Purpose of the Study:
- To report a novel FRET effect between Pacific Blue (PB) and FITC fluorochromes.
- To investigate the cause of an unexpected signal on the V550 (Krome Orange; KrO) detector in T cell analysis.
- To highlight the potential for FRET to cause misleading results in multicolor flow cytometry panel design.
Main Methods:
- Fresh whole blood was stained with a panel including anti-CD3-PB, CD8-KrO, Vbeta FITC, Vbeta PE, and CD4 AA750.
- Cells were fixed, treated for erythrolysis, washed, and analyzed on a DxFlex cytometer.
- Data analysis was performed using Kaluza software, including spectral analysis of fluorochromes.
Main Results:
- An unexpected CD8dim-KrO (V550) signal was observed on all Vβ FITC positive T cells when excited by a violet laser.
- This FRET effect between PB and FITC was confirmed by its dependence on the presence of both anti-CD3-PB and anti-Vβ-FITC antibodies.
- The FRET was also observed with anti-TCR gamma delta-FITC but not with other FITC-labeled antibodies not associated with the CD3-TCR complex.
Conclusions:
- A FRET effect between Pacific Blue (PB) and FITC, excited by the violet laser, has been described for the first time.
- This PB-FITC FRET can lead to misleading signals, particularly when labeling closely related markers like CD3 and TCR Vβ.
- Careful consideration of FRET is crucial during multicolor flow cytometry panel design to avoid erroneous interpretations.
Abstract:
Multiple immunolabeling introduces high risks of interferences between fluorescences. As an example, in analyzing T cell clonality, we recently reported a fluorescence resonance energy transfer (FRET) effect providing an unexpected signal on B770 (PE-Cy7) detector, on the Vβ-PE positive CD3 APC-Alexa750+ T cell subsets. Here, we report another FRET effect produced by the violet laser in Vβ-FITC positive CD3-Pacific Blue (PB) T cells providing signal on V550 (Krome Orange; KrO) detector. The study was performed on fresh whole blood, labeled with anti-CD3-PB, CD8-KrO, Vbeta FITC, Vbeta PE, CD4 AA750 then fixed, treated for erythrolysis, and washed before analysis on DxFlex cytometer from Beckman Coulter. Data were analyzed using Kaluza software. Using this panel, we repeatedly observed an added CD8dim-KrO (V550) cell population on all Vβ FITC positive T cells. The unexpected green signal excited by the violet laser was still observed after removing anti-CD8-KrO (FMO) but disappeared where either anti-CD3-PB or anti-Vβ-FITC was removed. The effect was also observed with an anti-TCR gamma delta-FITC labeling, but not with another FITC labeled antibody targeting a protein out of the CD3-TCR complex. The analysis of fluorochrome spectra confirms that PB emission and FITC excitation spectra partly overlap. This observation clearly reminds users that FRET can give misleading results in case of labeling of very close markers with complementary fluorochromes. This risk has to be considered in panel design. These observations clearly highlight the potential for FRET to give misleading results in cases where very close markers are labeled with complementary fluorochromes. This risk must be considered when designing panels. To our knowledge, this is the first description of a FRET between PB and FITC as acceptor thus excited by the violet laser.
