Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5

Abstract

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488nm. Mouse IgG, monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG, anti-PE using F(ab'), fragments of monoclonal rat anti-mouse IgG,. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/ Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.

A deep profiler's guide to cytometry

“…In the late 1980s (Figure 1), the remarkable ability of PE to absorb and transfer energy to other fluorescent molecules was recognized and exploited in order to produce an array of tandem dyes (e.g., PE-Texas Red, PE-Cy5, PE-Cy5.5, PE-Cy7)[8, 9]. In the 1990s, APC-based tandem dyes were synthesized[9], and a large spectrally-resolved series of small organic dyes (known as the Alexa dyes) became commercially available[10].…”

Detection of minimal residual disease in pediatric acute lymphoblastic leukemia

“…From the 1980s, the capacity of several molecules to absorb and transfer energy to other fluorescent molecules was exploited to produce tandem dyes such as PETexas Red, PE-Cy5, PE-Cy5.5, PE Cy7, and Alexa (27,28), thus many laboratories were soon routinely performing four-color experiments. Further advances rapidly led to the possibility of studying a great number of functionally distinct lymphocyte populations in human peripheral blood by the simultaneous detection of six to eight different cell surface antigens (29).…”

Telomere shortening in leukocyte subpopulations from baboons

“…#B30111, BDH Inc.) with 10 g/ml phycoerythrin (PE)-labeled human anti-CD45RA antibody (8d2; ref. [39]) and with 0.3 g/ml PE-labeled human anti-CD20 antibody (L26, kindly provided by Dr. Andre van Agthoven, Beckman Coulter Immunotech, Fullerton, CA). Both antibodies were previously found to cross-react with cells from various monkeys [40,41].…”