Selective single cell isolation for genomics using microraft arrays

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Cell killing by Frog Virus 3: Evidence for cell killing by single viral particles or single viral subunits

Cell killing by Frog Virus 3 was assayed after infection of chinese hamster ovary cells under non permissive conditions for virus multiplication. The kinetics of the loss in the efficiency of colony formation as a function of the virus multiplicity indicated that infection of a cell with a single viral particle brought about cell death. About 15 percent of the cells exhibited transient resistance to killing by single viral particles. Treatment of cells with proteins solubilized from Frog Virus 3 also resulted in cell killing with one hit kinetics thus implying that the interaction with a single viral subunit sufficed to entail cell death.     

Determination of dopamine in single rat pheochromocytoma cell by capillary electrophoresis with amperometric detection

A method is described for the direct identification of dopamine in single cultured rat pheochromocytoma cell by capillary electrophoresis (CE) with amperometric detection. The separation and detection conditions were optimized. The dopamine in single cell analysis was identified based on the migration time of standard dopamine and internal standard (epinephrine). The amount of dopamine in a single cell ranged from 0.29 to 1.28 fmol.     

The application of different cell lines and virus strains for detection of immunofluorescence rubella antibodies.

Fluorescence antibodies (FA) were titrated in human sera against antigens synthesized in four different infected cell lines: SIRC, RK-13, Vero and BHK-21. The cells infected in suspension by rubella virus attained the optimal concentration of fluorescence staining antigen earlier, fluorescence was more intensive and the titers of the tested human sera were slightly higher than in monolayer cell cultures. FA titers were high and they were correlated with the antibody titers obtained by hemagglutination-inhibition test. Some practical implications of these findings are discussed.     

mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

Background  

The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.     

piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics

Transposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the alpha1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4delta or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4delta/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila.     

Specific detection of cell surface-displayed human melanocortin 4 receptors with antibodies generated in mice.

The melanocortin-4 receptor (MC-4R) is a 7-transmembrane protein, which is involved in the central regulation of appetite and obesity. Despite the great interest in this protein, tools for detecting this molecule (as expressed on the cell surface in its native state) have been unavailable. Radioactive- or otherwise labeled ligands showed low receptor specificity to this particular melanocortin receptor isotype. Also, the antibodies were only available for epitopes that were displayed in the cytoplasm. To produce antibodies that enable the detection of this receptor (as expressed on the cell surface without disruption of the target cells), a candidate epitope was selected from the extracellular domains by a computer-aided analysis of the IC-4R secondary structure. This particular region was then recombinantly expressed in E. coli. Immunization of BALB/c mice with the recombinant proteins induced a specific immune reaction, which resulted in the production of MC-4R-specific antibodies. Enzyme-linked immunosorbent assays confirmed the specificity of these antibodies. To examine whether this tool also reacts with native cell surface-displayed MC-4R, HEK-293 cells were transfected with the human MC-4R cDNA. They were analyzed with these antibodies using Western blot and flow cytometry. Specificity and exclusion of cross-reactivity of these antibodies to other MC receptors were further confirmed by an immunofluorescence analysis of the HEK-293 cells that were transfected with other MC receptor isotypes. It is evident that with the availability of this tool, studies on the cell- and tissue-specificity, as well as the regulation mechanism of the MC-4 receptor, will be largely facilitated.     

Cell type discovery and representation in the era of high-content single cell phenotyping

Background

A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology.

Results

In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including “context annotations” in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations.

Conclusion

The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new technologies and support the data representation needs of the research community. The proposals enumerated here will serve as a useful starting point to address these challenges.

     

Effect of focus on cell detection and recognition by the Cell Analyzer

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.     

Use and detection of nonradioactive iodine-labeled antibodies for immunoassay

Traditionally radioactively labeled iodine has been used as a reporter group for the detection of antibodies in immunoassay. We have recently developed a microassay system for the detection of very low concentrations of iodide which eliminates the need for the use of radiolabeled iodine (O'Kennedy, R. et al. 1989, Anal. Biochem. 179, 138-144). The successful application of this assay for the detection of mouse IgG is described. The performance of this system compared with enzyme immunoassay was evaluated.     

Preparation of hybrid antibodies with mouse IgG and ferritin specifities for the immune electron microscopic detection of cell membrane antigens]

Use of hybrid antibodies with one specifity for mouse-IgG and the other one for ferritin is particularly suitable for immune electron microscopical detection of cell surface antigens. Preparation of antibodies of such kind is described, whereby the method introduced by HMMERLING et al. is varied within some steps of preparation. These variations are discussed here. Activity of produced hybrid antibodies is demonstrated by labeling the THY 1.1. antigen on the cell surface of the thymocytes of the mouse. The advantages of utilizing the hybrid antibodies in comparison with known immune electron microscopical techniques are an excellent location of the antigens, the possibility of using distinct particles for labeling, the application of a multiple labeling, and the fact that investigations by both the transmission and the scanning electron microscope can be carried out by means of the same preparation of hybrid antibodies.     

基于连接酶—ELISA反应的单核苷酸多态性分型新方法

随着大量与人类疾病和药物治疗相关的单核苷酸多态性（Single-nucleotide polymorphism,SNP）的发现,出现了多种SNP分型检测的方法和技术。然而,大多数方法由于受限于检测灵敏度低或对检测设备和实验条件要求较高,不适宜于在一般实验条件下进行常规临床检测。通过建立一种基于连接酶-ELISA的SNP快速分型新方法,以非小细胞肺癌个体化治疗中,酪氨酸激酶抑制剂药物的生物标记基因—表皮生长因子受体基因（EGFR）为检测对象,对EGFR,c.2573T〉G（L858R）,EGFR,c.2582T〉A（L861Q）和EGFR,c.2155 G〉T（G719C）3个SNP位点进行了突变检测。经过18~28个循环的PCR扩增,能够通过琼脂糖凝胶电泳和ELISA反应,根据电泳条带的有无和ELISA显色值清晰判断检测位点的基因型,并且能够从混合等位基因样本中检测出5%的突变型等位基因。结果表明,方法具有较高的特异性和灵敏度,适合于在常规实验条件下从不均一的样本中进行突变等位基因的检测。     

Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo.     

What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.     

Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution

By using a fine oil-filled glass microcapillary mounted on a micromanipulator, the solutes of individual plant cells can be sampled. These samples can then be analysed using a range of physical and chemical methods. Hydrostatic pressure (cell pressure probe), osmotic pressure (picolitre osmometer), organic solutes (enzyme-linked fluorescence microscope spectrometry or capillary electrophoresis), inorganic solutes (X-ray microdroplet analysis or capillary electrophoresis), (14)C (mass spectrometry), proteins (microdroplet immunoblotting), and mRNA (rt PCR) have been measured. Collectively, the battery of techniques is called single cell sampling and analysis (SiCSA) and all of the techniques have relevance to the study of plant metabolism at the resolution of the individual cell. This review summarizes the techniques for SiCSA and presents examples of applications used in this laboratory, in particular those relating to cell metabolism.