Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry

A method is presented for encapsulating single microbial cells in small spheres suitable for analysis and sorting by flow cytometry. The entrapped cells are able to multiply and form colonies contained within their respective microspheres. The system is based on ejecting the cells suspended in a gellable liquid through an orifice vibrating at ultrasonic frequencies, thus shearing the cell-containing jet into uniform droplets. When low-melting-temperature agarose was used, the droplets could be gelled into solid spheres during flight by appropriately directed colling air streams. This gelling was accompanied by significant dehydration, resulting in a twofold decrease in bead diameter and a corresponding increase in agarose concentration. Nevertheless, the microbeads obtained were highly uniform and had diameters which could be precisely controlled in the range of 10 to 40 microns. A variety of bacterial and yeast species were entrapped in agarose beads by using this system. In all cases the cells were able to develop into microcolonies containing as many as several hundred cells. This system enables one to apply the powerful method of flow cytometry to the analysis and sorting of whole microbial colonies. Potential applications of this technology in various areas of microbiology are considered.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.     

Multiparameter analysis and sorting of mammalian cells

An improved multisensor cell sorting instrument for quantitative analysis and sorting of cells has been developed. Cells stained with fluorescent dyes enter a flow chamber where cell volume, fluorescence, and light scatter sensors simultaneously measure multiple cellular properties. Cells then emerge in a liquid jet that is broken into uniform liquid droplets. Sensor signals are electronically processed in one of several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a multichannel pulse-height analyzer. Processed signals activate cell sorting according to preselected parametric criteria by electrically charging droplets containing cells and electrostatically deflecting them into collection vessels. Illustrative examples of multiparameter cell analysis and sorting experiments using a model mouse tumor cell system, human and animal leukocytes, and cultured mammalian cells are presented.     

Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ～25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.     

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.     

The active human gut microbiota differs from the total microbiota

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around 75% of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.     

Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of DNA typing after sexual assault.

BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. METHODS: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. RESULTS: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. CONCLUSIONS: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.     

Gel microdroplet technique leaving microorganisms alive for sorting by flow cytometry

Fluorescent labels used to disclose cellular function and the like are generally needed for cytometric analysis, but suitable ones are not always available. Thiamin, an example of such a label, was used as a model in this study. Thiamin in cells of bakers' yeast can be chemically converted to thiochrome, which fluoresces strongly, but the reaction kills cells not protected inside a gel microdroplet (GMD). Our new procedure for preparation of a small amount of GMDs uses a glass filter with micropores uniform in size to make an emulsion of molten agar in mineral oil that is then chilled for gelation of the agarose. Cells were suspended and mixed with the warm agarose, which was used to make GMDs. Cells were grown into microcolonies in the GMDs, which were then treated to convert thiamin to thiochrome. The thiochrome in microcolonies in single GMDs could be detected by flow cytometry. Plating showed that enough cells survived for this method to be used for screening. This technique can be used for all applications of GMDs.     

Gene transfer by electroporation, lipofection, and DEAE-dextran transfection: compatibility with cell-sorting by flow cytometry.

The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were met with COS monkey kidney cells that express a recombinant glutathione S-transferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony-forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20-22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection with DEAE-dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25-40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.     

Kinetic aspects of the bioconversion of L-tyrosine into L-DOPA by cells of Mucuna pruriensL. Entrapped in different matrices

Plant cells of Mucuna pruriens L. entrapped In calcium alginate, calcium pectinate, agarose, or gelatine were able to convert L-tyrosine to L-DOPA, which was released Into the medium. Michaelis-Menten kinetics could be applied on the entrapped cells, based on the measurement of initial rates of L-DOPA production. The calculated apparent affinity constants were comparable with the affinity constants obtained with enzyme preparations. Comparison of the apparent maximum rate of bioconversion of the entrapped cells and the maximum rate of bioconversion of a derived cell homogenate indicated that the systems were not operating optimally. Measurement of the effective diffusion coefficients of L-tyrosine pointed out that this substrate could diffuse freely into the matrices. From the initial rates of bioconversion and the effective diffusion coefficients, the observable modulus was calculated for each system. The obtained values confirmed that the diffusional supply rate of L-tyrosine was not the limiting factor. For oxygen, which was utilized for byconversion as well as for cell respiration, the calculated observable moduli was directed toward strong oxygen transfer limitations. The values found for the oxygen consumption indicated that the entrapped cells remained partly or totally viable in the four matrices tested. Based on the highest viability and the highest rates of bioconversion, it was concluded that alginate-entrapped cells of M. pruriens formed the most suitable biocatalytic system for the production of L-DOPA from L-tyrosinre.     

Combination effects of complement regulatory proteins and anti-complement polymer

We previously reported the development of a "cytomedicine" that consists of cells trapped in alginate-poly-L-lysine-alginate (APA) microcapsules and agarose microbeads. The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system. However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low. Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system. We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine. Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack. These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.     

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.     

New possibilities for studying complex macromolecular structures of the cell by encasing cells in polyacrylamide microspheres]

A method is proposed for incorporation of mammalian cells into spheres permeable for protein molecules, the dimensions of these spheres not overcoming those of the cells. The method permits fractionation of the cells, preserving them as discrete units resistant to mechanical and hydrodynamic destruction in the course of routine laboratory manipulations, as well as using flow cytometry for their analysis. With this approach, the flow cytometric measurements were made of DNA being shielded in the chromatin complex which prevents from binding fluorescent dyes with low-molecular weights.     

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.     

Construction of low melting point agarose emulsion PCR amplification complex gene sequence

Emulsion polymerase chain reaction, an effective amplification, can make millions of templates could be individually amplified within a single tube. Here we constructed and improved a low melting point agarose-emulsion method to promote the specific sequences amplification effectively. Artificial Lactobacillus Plasmid as template was amplified and clear fluorescence images of the agarose beads were obtained. The Real-time PCR data showed that agarose-emulsion PCR clearly indicated that DNA can be amplified in agarose droplets. Overall, our study effectively overcame the difficulty of formation of uniform emulsion droplets, negative effect on recombination of homologous regions of DNA and generation of void emulsion droplets. This method increases the accuracy with amplification, reduces the influence of uncertainties and provides the reliable data for further experiment.     

Transmission electron microscopy of small numbers of sorted cells

A new method that allows the transmission electron microscopic examination of as few as 1 x 10(4) cells obtained by flow cytometric sorting is described. The approach involves "sandwiching" fixed cells in an agarose case by a microcentrifugation system consisting of small-diameter cell-centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.     

Early chick embryonic cells can form clones in agarose cultures

Early chick embryonic cells prior to the formation of the primitive streak, have been cultured in a two-layer soft-agarose system. Single, primary cells when grown in this system were capable of producing colonies ranging in size from 30 to 100 cells. The plating efficiency varied between 1 and 5% and the colonies remained viable for about 2 weeks. We believe this is the first report of normal, non-passaged cells which show anchorage-independent growth properties by forming colonies in a standard agarose culture in the absence of additional factors. The importance of being able to use normal monoclonal embryonic cell populations in studying early developmental processes is also discussed.     

Isolation of clones of hamster embryo cells transformed by the bovine papilloma virus

Primary hamster embryo cells infected with bovine papilloma virus (BPV) or treated with BPV DNA-calcium phosphate precipitates showed striking morphological alterations characteristic of transformed cells. Long, spindle-shaped cells grew into dense foci, eventually overgrowing monolayers of normally shaped cells. Samples of these cells were tested for anchorage-independent growth in dilute agarose medium. Cells were able to grow in agarose to form colonies which, when removed from agarose and transferred to liquid medium, established clones. Mockinfected cultures inoculated with plain medium displayed normal cell morphology and growth properties. This is the first report of BPV-transformed cells demonstrating anchorage-independent growth in agarose and the establishment of BPV transformed clones.     

微生物活细胞快速检测的新技术研究

微生物活菌分为繁殖能力的活菌和有代谢活动但没有繁殖能力的活菌。传统的平板计数方法无法对有代谢活动但没有繁殖能力的活菌进行准确定量分析。本文综述了微生物活细胞检测新技术的研究进展,包括以PCR为基础的新技术和荧光活化细胞分选术与流式细胞术结合的新技术。