Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

BackgroundConventional immuno‐based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody‐fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10–15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence‐intensity multiplexing. By varying the Zenon labeling reagent‐to‐antibody molar ratio, the fluorescence intensity of the antibody‐labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno‐based multiplexed flow cytomety assay.MethodsLymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence‐intensity multiplexing analysis. Conventional analysis measures a single antibody‐fluorophore per photomultiplier tube (PMT). Fluorescence‐intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent‐antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor®488 Mouse IgG1 labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R‐Phycoerythrin (R‐PE) Mouse IgG1 or IgG2b labeling reagents.ResultsThe lymphocyte immunophenotyping results from fluorescence‐intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis.ConclusionsSimultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection‐channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2‐ or 3‐detection‐channel flow cytometers. For typical biological samples, approximately 2–4 cellular targets per detection channel can be resolved using this technique. © 2004 Wiley‐Liss, Inc.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in the treatment of human immunodeficiency virus since the introduction of azidothymidine (AZT) in 1987. However, none of the current therapies can completely eradicate the virus, necessitating long-term use of anti-retroviral drugs to prevent viral re-growth. One of the side effects associated with long-term use of NRTIs is mitochondrial toxicity stemming from inhibition of the mitochondrial DNA (mtDNA) polymerase gamma, which leads to mtDNA depletion and consequently to mitochondrial dysfunction. Here we report the use of fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI 2',3'-dideoxycytidine (ddC). These techniques are amenable to both microscopy and flow cytometry, allowing analysis of populations of cells on a single-cell basis. We show that, as mtDNA depletion progresses, a mosaic population develops, with some cells being depleted of and others retaining mtDNA. These techniques could be useful as potential therapeutic monitors to indicate when NRTI therapy should be interrupted to prevent mitochondrial toxicity and could aid in the development of less toxic NRTIs by providing an assay suitable for pharmacodynamic evaluation of candidate molecules.

Introduction:In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton‐ion lasers on stream‐in‐air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low‐level expression of Cyan Fluorescent Protein (ECFP) in CFP‐YFP FRET applications. In this follow‐up study, we have tested more powerful VLDs emitting at 55 mW, and a beam‐merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet‐excited fluorochromes, including CFP.Methods:A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream‐in‐air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet‐excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet‐excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel‐coupled cuvette flow cytometer was used as a sensitivity baseline.Results:The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower‐power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source.Conclusions:While 55 mW laser sources performed adequately in the stream‐in‐air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems. © 2006 International Society for Analytical Cytology

Flow cytometry and fluorescence microscopy both provide single-cell analysis using different but complementary sets of data, essentially population-based target intensities versus target morphology in relatively small sample sizes. Both approaches generally employ optical filters to analyze fluorescence emissions, and have to overcome some of the same physical limitations, including spectral overlap of dyes and the dynamic range limits of measuring systems. Some of the technical challenges differ: dye photostability is more critical to microscopy; creating suspensions from adherent cells can impact flow cytometric analysis. With extensive image acquisition and processing, the microscopist may arrive at quantitative data. However, the cooperative use both flow cytometry and microscopy can provide more robust interrogation of biological phenomena.A number of examples will be presented to illustrate both the complementarity that microscopy and flow cytometry perspectives bring to new applications and ultimately, biological questions. They also illustrate the constraints to migrating assays between platforms. Viability assays based on membrane integrity or esterase activity generally provide bright, well-resolved signals easily read by microscopy or flow cytometry (Figures 1-2). Microscopy has helped verify mitochondrial specificity and responsiveness with mitochondrial function assays that can be quantified by flow cytometry. However, assays that depend on translocation rather than overall intensity change, such as loss of cytochrome c from mitochondria, are better performed by microscopy. Flow cytometry can readily quantify live cells in various stages of cell cycle using a number of cell-permeant DNA dyes; microscopy confirms nuclear labeling and also indicates that some of these dyes may have adverse morphological affects. Kinetic studies, such as calcium response, can be accomplished on individual cells by microscopy, and easily quantified on populations by flow cytometry. Morphological information, as with organelle structure, is readily available through microscopy, but flow cytometry may provide little useful information about morphology. One example of complementarity between microscopy and flow cytometry is provided by analysis of mitochondrial DNA (mtDNA) depletion. Microscopy has demonstrated the decrease of mtDNA-encoded COXI, including mosaicism, in individual fibroblasts following progressive exposure to anti-retroviral drugs. Flow cytometry has provided rapid population statistics to support the visual results. 1,2Fluorescence microscopy is well suited to the resolution of cell and tissue architecture, and to following kinetic and trophic responses in single cells. Flow cytometry rapidly quantifies small differences between cell populations using statistically-significant numbers of events. Flow cytometry can represent a "black box" when looking at the magnitude of a population response; fluorescence microscopy can help verify that measured results represent meaningful biological effects.