Fluorescence intensity calibration for immunophenotyping by flow cytometry

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional 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. METHODS: Lymphocyte 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 IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous 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.     

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.     

Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.     

Enrichment for submitotic cell populations using flow cytometry

BACKGROUND: One of the most dramatic events during the course of the mammalian cell cycle is mitosis, when chromosomes condense and segregate, the nuclear envelope breaks down, and the cell divides into two daughter cells. Although cells undergoing mitosis are cytologically distinguishable from nonmitotic cells, few molecular markers are available to specifically identify mitotic cells, especially cells within different stages of mitosis. METHODS: We applied the flow cytometric method of Juan et al. (Cytometry 32:71-77, 1998) to obtain cells with various levels of the molecular markers cyclin B1 and phosphorylated histone H3; fluorescence microscopy was then used to identify sorted cells in different stages of mitosis. RESULTS: We observed the substantial enrichment of submitotic cell populations. CONCLUSIONS: This method represents an effective approach to obtain an enriched population of submitotic cells without the use of drug treatments or prior synchronization.     

基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础,以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L,反应时间为60 min。当菌浓在104 107cells/mL范围时,检测值可信度较高,可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测,变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。     

Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.     

Clinical application of four and five-color flow cytometry lymphocyte subset immunophenotyping

A 1995 survey of clinical flow cytometry laboratories in the United States determined that 63% of clinical laboratories used one or two-color, 33% used three-color, 4% used four-color, and none used five-color panels. We show the feasibility and advantages of acquiring routine clinical four-color, six-parameter and five-color, seven-parameter analysis on blood samples derived from a pediatric population. The panels were evaluated by comparing the following cell characteristics: size, internal structure, and up to five distinct fluorochrome-conjugated antibodies to cell surface antigens: CD3, CD16+CD56, CD19, CD8, and CD4. These samples were processed on a commercially available instrument without any special modifications. A comparison of two-color and four-color as well as four-color and five-color panel analysis showed no statistical difference between the groups. We propose that the five-color, single-tube panel will (1) eliminate the need for isotype controls; (2) the relative proportions of lymphocyte subpopulations may be used to validate the operator-defined window, replacing CD45; (3) eliminate the need to run a common factor, in order to establish and maintain a reproducible lymphocyte window between tubes; and (4) create a more complete clinical picture by generating 32 unique, mutually exclusive phenotypes (permutations). Our results show that it is feasible to acquire and integrate seven-parameter data. This may be a powerful tool for immunophenotyping cells in a modern clinical diagnostic cytometry laboratory.     

Time interval gating for analysis of cell function using flow cytometry.

We propose a method which significantly shortens the time required for both the collection and analysis of data derived from multiple sample, flow cytometric kinetic assays. We have defined the term Time Interval Gating (TIG) to describe this method. TIG effectively allows one flow cytometer to concurrently monitor several samples over the course of a kinetic assay. Data for all samples are stored in a single FCS 2.0 compatible listmode data file which we refer to as the TIG data file. TIG is adaptable to most commerical flow cytometers. Standard listmode analysis software can be used to analyze the TIG data files and correlate any combination of tubes and/or time intervals from the assay. Results for the entire assay can be displayed on a single two parameter plot. This paper describes how TIG is applied to neutrophil oxidative burst measurement using a standard EPICS Elite flow cytometer. In this assay, 11 samples were each monitored for 30 min to identify the extent to which volatile organic chemicals (VOCs) inhibited the oxidation of DCFH in stimulated neutrophils. TIG makes the oxidative burst assay practical for high volume screening by reducing the overall flow cytometer and analysis time required by a factor of ten. In addition, TIG provides an organized approach to managing data acquisition on instruments equipped with automated sampling systems.     

A fast cell sampler for flow cytometry

A simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 microliter, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever-operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.     

Purification of human fetal hippocampal neurons by flow cytometry for transplantation

We have established primary cultures, highly enriched in neurons, from the hippocampus of human fetal brains at 20-23 gestational weeks. More than 80% of cells were viable when seeded. Neurons were isolated from primary cultures by flow cytometry to a high degree of purity, as demonstrated by immunocytochemical staining. FACS scanning analysis using a DNA-staining dye showed that hippocampal neurons did not divide in culture. To demonstrate that FACS-sorted neurons can be transplanted and integrated into the host brain, neuron-enriched primary culture from human fetal striatum was infected with a viral-mediated vector containing a reporter gene, beta-galactosidase. Striatal neurons were subsequently purified by flow cytometry and transplanted into the striatum of rats. Following transplantation, the rat brains were processed for beta-galactosidase histochemistry and electron microscopy. Beta-galactosidase expression indicates that transplanted human neurons survived in the host and were metabolically active. The transplanted neurons received synaptic inputs, as judged from the presence of presynaptic terminals on their surface. Our study demonstrates connectivity between transplanted human fetal primary neurons and host tissue at the ultrastructural level. Our results support the feasibility of ultimately transplanting neurons into humans as a possible treatment for recovery of the nervous system (e.g., neurodegenerative diseases).     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

Automated flow cytometry for monitoring CHO cell cultures

Flow cytometry has been used to accurately monitor cell events that indicate the spatio-temporal state of a bioreactor culture. The introduction of process analytical technology (PAT) has led to process improvements using real-time or semi real-time monitoring systems. Integration of flow cytometry into an automated scheme for improved process monitoring can benefit PAT in bioreactor-based biopharmaceutical productions by establishing optimum process conditions and better quality protocols. Herein, we provide detailed protocols for establishing an automated flow cytometry system that can be used to investigate and monitor cell growth, viability, cell size, and cell cycle data. A method is described for the use of such a system primarily focused on CHO cell culture, although it is foreseen the information gathered from automated flow cytometry can be applied to a variety of cell lines to address both PAT requirements and gain further understanding of complex biological systems.     

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.     

Mammalian cell culture scale-up and fed-batch control using automated flow cytometry

Detailed knowledge of mammalian cell culture proliferation kinetics is important to determine fed-batch strategies for industrial bioreactor operations. In particular, predicting the end of exponential proliferation in batch culture is a critical process parameter during culture scale-up. Using automated flow cytometry we show that an increase in the non-viable sub-population in CHO cell culture can predict the onset of stationary phase by approximately 40 h. This enables a completely automated culture scale-up process as well as a reliable and reproducible control of fed-batch additions during culture expansion. It is shown that the automated scale-up results in a significantly higher total cell count in the reactor than manual scale up initiated in stationary growth phase. During individual, subsequent culture expansions, a significant variation in the proliferation rate was observed despite control of bulk culture parameters. Thus, automated flow cytometry is critical to uncovering useful process parameters that enable new control strategies. Such improved process supervision derived from knowledge-based data analysis is central to the FDA's Process Analytical Technology (PAT) initiative and is expected to result in better and higher quality products.