FRET causing misleading signal from fluorescein excited by the violet laser in flow cytometry.
FITC
FRET
Pacific Blue
flow cytometry
phycoerythrin
violet laser
Journal
Cytometry. Part A : the journal of the International Society for Analytical Cytology
ISSN: 1552-4930
Titre abrégé: Cytometry A
Pays: United States
ID NLM: 101235694
Informations de publication
Date de publication:
09 2023
09 2023
Historique:
revised:
21
06
2023
received:
27
03
2023
accepted:
20
07
2023
medline:
21
9
2023
pubmed:
8
8
2023
entrez:
8
8
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37552188
doi: 10.1002/cyto.a.24780
doi:
Substances chimiques
Fluorescein
TPY09G7XIR
Fluorescent Dyes
0
Fluorescein-5-isothiocyanate
I223NX31W9
CD3 Complex
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
732-735Informations de copyright
© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
Références
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