Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure.     

Analysis of virus-infected cells by flow cytometry

Flow cytometry has been used to study virus-cell interactions for many years. This article critically reviews a number of reports on the use of flow cytometry for the detection of virus-infected cells directly in clinical samples and in virus-infected cultured cells. Examples are presented of the use of flow cytometry to screen antiviral drugs against human immunodeficiency virus (HIV), human cytomegalovirus, and herpes simplex viruses (HSV) and to perform drug susceptibility testing for these viruses. The use of reporter genes such as green fluorescent protein incorporated into HIV or HSV or into cells for the detection of the presence of virus, for drug susceptibility assay, and for viral pathogenesis is also covered. Finally, studies on the use of flow cytometry for studying the effect of virus infection on apoptosis and the cell cycle are summarized. It is hoped that this article will give the reader some understanding of the great potential of this technology for studying virus cell interactions.     

Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay

Melittin, from the honeybee venom, is a membrane active protein, whose cytotoxicity to human endothelial cells has not been described yet. In this work, we studied its time-dependent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Since HUVECs grow in culture as adherent cells, suspension of cells is required before measuring cytotoxicity with a haemocytometer or flow cytometry. Therefore, we also tried to discover whether the result of cytotoxicity tests of melittin is influenced by the preparation of the cell suspension. For this purpose, we compared the results of haemocytometer-based trypan blue assay and flow cytometry using 7-aminoactinomycin D (7-AAD) with results of fluorescence microscopy using 7-AAD and 4', 6-diamidino-2-phenylindole (DAPI). Melittin over 60 min exposure evoked a rapid decline in the survival of HUVEC. After 60 min exposure to melittin, the phase contrast microscopy demonstrated massive necrosis in the remaining attached cells. Fluorescence microscopy detected both viable and non-viable cells in adequate proportions at all exposure times, whereas haemocytometer-based assay and flow cytometry highly underestimated the percentage of non-viable cells or even failed to detect any dead cells. Our data clearly indicate that the induction of large-scale damage to adherent endothelial cells by melittin results in a loss of the majority of necrotic cells during sample preparation for flow cytometry or a haemocytometer-based assay. In the case of adherent cell culture, therefore, fluorescence microscopy was shown to be a more appropriate method for quantitative analysis of cell death caused by a fast-acting cytolytic toxin such as melittin.     

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.     

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。     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry¹ offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture⁵. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.     

Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.     

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.     

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。     

Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

Abnormalities in lymphocyte signaling cascades are thought to play an important role in the development of autoimmune disease. However, the large amount of cellular material needed for standard biochemical assessment of signaling status has made it difficult to evaluate putative abnormalities completely using primary lymphocytes. The development of technology to employ intracellular staining and flow cytometry to assess the signaling status of individual cells has now made it possible to delineate the perturbations that are present in lymphocytes from patients with autoimmune disease. As an example, human B cells from the Ramos B cell line and the periphery of systemic lupus erythematosus (SLE) patients or normal nonautoimmune controls were assessed for activation of the NF-kappaB and mitogen activated protein kinase (MAPK) signaling cascades by intracellular multiparameter flow cytometric analysis and biochemical Western blotting. In combination with fluorochrome conjugated antibodies specific for surface proteins that define B cell subsets, antibodies that recognize activated, or phosphorylated inhibitors of kappaB (IkappaB) as well as the extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) or p38 MAPKs were used to stain fixed and permeabilized human B cells and analyze them flow cytometrically. Examination of the known signaling pathways following engagement of CD40 on human B cells confirmed that intracellular flow cytometry and Western blotting equivalently assay CD154-induced phosphorylation and degradation of IkappaB proteins as well as phosphorylation of the MAPKs ERK, JNK and p38. In addition, B cells from the periphery of SLE patients had a more activated status immediately ex vivo as assessed by intracellular flow cytometric analysis of phosphorylated ERK, JNK and p38 when compared with B cells from the periphery of normal, nonautoimmune individuals. Together, these results indicate that multiparameter intracellular flow cytometric analysis of signaling pathways, such as the NF-kappaB and MAPK cascades, can be used routinely to assess the activation status of a small number of cells and thus delineate abnormalities in signaling molecules expressed in primary lymphocytes from patients with autoimmune disease.     

Analysing phosphorylation-based signalling networks by phospho flow cytometry

Analysis of signalling events by classical biochemical approaches is limited as the outcome is an averaged readout for protein activation of a single protein within a cell population. This is a clear restriction when addressing signalling events in mixed populations or subpopulations of cells. By combining flow cytometry with a panel of phosphospecific antibodies against several signal molecules simultaneously in a multi-parameter phospho flow cytometry analysis it is possible to obtain a higher level of understanding of the signal transduction dynamics at a single cell level. In addition, analysis of mixed cell populations makes it possible to study cells ex vivo in a state more closely resembling the in vivo situation. The multimeric analysis yields information on combinations of signals turned on and off in specific settings such as disease (signal nodes) that can be used for biomarker analysis and for development of drug screening strategies. Prostaglandin E₂ (PGE₂) is known to signal through four G-protein coupled transmembrane receptors, EP1-4, activating a multitude of potential signalling pathways. The analysis of the PGE₂ signalling network elicited by activation of the four EP receptors in lymphoid cells revealing several signalling nodes is reviewed as an example.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

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.     

Flow cytometric evaluation of adhesion of Streptococcus pyogenes to epithelial cells

The precise roles of various surface molecules in the attachment of Streptococcus pyogenes to host epithelia are currently unclear. A flow cytometry assay that facilitates the analysis of the kinetics of S. pyogenes adhesion to epithelial cells was developed. Dose- and time-dependent adhesion isotherms with both buccal epithelial cells (BECs) and Hep-2 cells as substrata were obtained. Although binding equilibrium is reached within 2 h on both cell types, saturation of binding sites on BECs is not achieved within a wide range of experimental conditions. This indicates a high degree of non-specific attachment to that cell type. Since no rinsing step is necessary when using flow cytometry to analyze adhesion, low-affinity associations were observable. This was confirmed by determining bacterial desorption rates early and late in the adsorption process. Binding irregularities were also easily detected since the cytometer records and displays data for up to 10,000 epithelial cells per time point. It is proposed to use this methodology to assign roles to particular surface molecules/characteristics during distinct phases of adhesion.     

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.     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

N-methylformamide effects on cell proliferation and antigenic pattern in HT-29 colon carcinoma cell line

The effects of the differentiating agent N-methylformamide (NMF) on cell proliferation and antigenic pattern of HT-29 colon carcinoma cells have been investigated. The cell line was cultured in the presence, or absence, of 1% NMF and tested for the above mentioned characteristics, both in vitro and after injection into nude mice. The percentage of cells in the various cell cycle compartments was estimated by flow cytometry. The presentation on the cell surface of molecules such as tumour associated antigens (TAAs), HLA class I molecules and epidermal growth factor receptor (EGF-R) was analysed by ELISA, flow cytometry and immunohistochemistry. Results demonstrate that NMF impairs HT-29 cell proliferation with a remarkable accumulation in the G0/G1 phases, as well as inducing a modification of the membrane antigenic pattern. The presence of NMF in the culture medium decreases the TAAs and EGF-R whereas HLA antigen maintains the same level of positivity in the two cell lines. These alterations are consistent with a different behaviour in vivo of the tumours originated from NMF treated and untreated cells. Tumours derived from NMF treated cells show a delay in the appearance and low levels of immunodetectable carcinoembryonic antigen (CEA) molecules.