Somatic cell genetics and flow cytometry

Human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescence on viable cells, the genes themselves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface.     

A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment

Currently, there is no standardized panel for immunophenotyping myeloid cells in mouse spleen using flow cytometry. Markers such as CD11b, CD11c, F4/80, Gr-1, Ly6C, and Ly6G have long been used to identify various splenic cell myeloid populations. Flow cytometry and fluorescence-activated cell sorting (FACS) analysis demonstrated that Ly6G/Ly6C markers are superior to Gr-1 for identifying splenic neutrophils, eosinophils, and subsets of monocytes/macrophages. Moreover, these experiments showed that F4/80 is not required for identifying these myeloid subsets and that many of the commercially available preparations of anti-F4/80 antibodies stain poorly for this antigen in spleen. Taken together, we have now developed an informative flow cytometry panel that can be combined with other cell markers to further delineate subpopulations of mouse splenic myeloid cells. This panel will be highly useful to investigators in the flow cytometry field, as there is a critical need to standardize the analysis of myeloid cell subsets.     

Recovery of RNA from flow-sorted fixed cells.

We describe a method for the preparation of RNA from ethanol-fixed cells, allowing analysis of the RNA from cells "frozen" in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations before RNA preparation, such as investigations of cell-cycle-regulated gene expression, which require the preparation of cells for cell-cycle flow analysis, and even for long-term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell-cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.     

Use of a lectin as an enterocyte-specific cell surface marker for flow cytometric analysis of isolated native small intestinal epithelial cells

Little use has been made of flow cytometry in evaluating small intestinal epithelial cells. Obtaining pure epithelial cell populations devoid of peripheral blood contaminants and intraepithelial lymphocytes contributes to the difficulties encountered in flow cytometry studies. We have investigated the use of lectins as enterocyte specific cell markers using lectin histochemistry, and have identified one lectin, UEA-1, which binds exclusively and specifically to intestinal epithelial cell brush border. Additionally, we have exploited that specificity using flow cytometry and FITC-UEA-1 to identify and separate native intestinal epithelial cells from a mixed cell population isolated by mechanical vibration. This fluorescent-lectin technique is a unique and simple method to identify native small intestinal epithelial cells in a mixed cell population; it may be exploited by flow cytometric sorting of a pure population for biochemical study or as an enterocyte specific label for surface receptor flow cytometric studies in the research or clinical setting.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

Integrating cytomics and proteomics

Systems biology along with what is now classified as cytomics provides an excellent opportunity for cytometry to become integrated into studies where identification of functional proteins in complex cellular mixtures is desired. The combination of cell sorting with rapid protein-profiling platforms offers an automated and rapid technique for greater clarity, accuracy, and efficiency in identification of protein expression differences in mixed cell populations. The integration of cell sorting to purify cell populations opens up a new area for proteomic analysis. This article outlines an approach in which well defined cell analysis and separation tools are integrated into the proteomic programs within a core laboratory. In addition we introduce the concepts of flow cytometry sorting to demonstrate the importance of being able to use flow cytometry as a cell separation technology to identify and collect purified cell populations. Data demonstrating the speed and versatility of this combination of flow cytometry-based cell separation and protein separation and subsequent analysis, examples of protein maps from purified sorted cells, and an analysis of the overall procedure will be shown. It is clear that the power of cell sorting to separate heterogeneous populations of cells using specific phenotypic characteristics increases the power of rapid automated protein separation technologies.     

Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting

Encapsulation of cells in agarose gel microdrops (GMDs) combined with fluorescence-activated cell sorting (FACS) has been used previously to analyze and recover specific mammalian, bacterial, and yeast cell populations. Recently, we have developed a method to enrich mixed bacterial populations for slow-growing microorganisms using the GMD Growth Assay combined with fluorochrome staining and flow cytometry. Here, we demonstrate the feasibility of using this experimental approach to detect clonogenic growth of individual bacteria within GMDs in less than 3 h and to separate subpopulations based on differential growth rates. We show that after sorting, organisms remain viable and can be propagated in culture for further analysis.     

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒（如细胞,细菌）的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来：功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

Analysis of apoptosis by laser scanning cytometry

Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.     

Isolation and visualization of Met-72-positive, metastatic variants present in B16 melanoma tumor masses

Metastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000-dalton glycoprotein (Met-72) on their cell surface (Kimura AK, Xiang J: J Nat Can Inst 76:1247-1253, 1986). Monoclonal antibodies (MoAb) directed against the Met-72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met-72-positive metastatic variants within tumor masses. Biotinylated anti-Met-72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)-streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met-72-positive cells were then cloned and reanalyzed after several weeks of in vitro expansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met-72 staining were positioned perivascularly and at the invading front of B16 melanoma tumors.     

Applications of flow cytometry sorting in the pharmaceutical industry: A review

The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures.     

Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.     

Monoclonal antibody isolation of type II pneumocytes

A monoclonal antibody that identifies a membrane molecule unique in rat lung for type II alveolar epithelial cells was used to isolate these cells from enzymatically dispersed lung cells by fluorescence-activated cell sorting. Although multistep physical separation techniques have permitted the isolation of large quantities of these cells and flow cytometry has been used by others to isolate lamellar body-containing cells, the application of this antibody-directed sorting has distinct advantages. Because the marker molecule is expressed on immature type II cells prior to the development of lamellar bodies, the antibody will also permit their isolation and study.     

Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed. Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream(X) system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.