ABSTRACT
Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.
Subject(s)
Cell Separation , Cytophotometry , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , Cells, Cultured , Cytophotometry/instrumentation , Humans , Jurkat Cells , Microfluidic Analytical Techniques/instrumentation , Particle Size , Surface PropertiesABSTRACT
Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.
Subject(s)
Electrophoresis, Capillary/instrumentation , Single-Cell Analysis/instrumentation , Animals , Cytophotometry/instrumentation , Cytophotometry/methods , Electrophoresis, Capillary/methods , Equipment Design , Humans , Microscopy/instrumentation , Microscopy/methods , Single-Cell Analysis/methods , SoftwareABSTRACT
INTRODUCTION: Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in many pathological settings. The aim of this two-site study was to evaluate the Sysmex XN Body Fluid mode (XN-BF) for cell analysis of CSF compared to light microscopy (LM). METHODS: Two hundred and seven consecutive CSF samples were analyzed in parallel with XN-BF and LM. The study also included the estimation of the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carry-over and linearity of XN-BF module. RESULTS: LoQ of white blood cells (WBC) was 3×106 cells/L; linearity was good and carry-over negligible. XN-BF parameters were compared to LM for the following cell classes: total cells, WBC, polymorphonuclear (PMN), and mononuclear (MN) cells. The bias ranged from 1.3 to 15.2×106 cells/L. The receiver operating characteristics curve analysis for WBC showed an area under the curve of 0.98, and the global diagnostic agreement was 95% at a cutoff of 5×106 cells/L. CONCLUSIONS: XN-BF provides rapid and accurate counts in clinically relevant ranges of CSF values, thus providing a valuable alternative to conventional LM analysis. However, microscopic review remains advisable in samples with abnormal cell counts or high fluorescent (HF-BF) cell parameter exceeding 5×106 cells/L.
Subject(s)
Cerebrospinal Fluid , Cytophotometry/instrumentation , Cytophotometry/methods , Leukocytes/pathology , Adult , Female , Humans , Leukocyte Count/instrumentation , Leukocyte Count/methods , MaleABSTRACT
Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells.
Subject(s)
Chromaffin Granules/metabolism , Electrochemical Techniques/methods , Exocytosis , Action Potentials , Animals , Chromaffin Granules/physiology , Cytophotometry/instrumentation , Cytophotometry/methods , Electrochemical Techniques/instrumentation , HumansABSTRACT
Cells respond to their mechanical environment by initiating multiple mechanotransduction signaling pathways. Defects in mechanotransduction have been implicated in a number of pathologies; thus, there is need for convenient and efficient methods for studying the mechanisms underlying these processes. A widely used and accepted technique for mechanically stimulating cells in culture is the introduction of fluid flow on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to fluid flow in culture. This device integrates with common lab equipment including routine cell culture plates and peristaltic pumps. Further, it allows the fluid flow treated cells to be examined with outcomes at the cell and molecular level. We validated the device using the biologic response of cultured UMR-106 osteoblast-like cells in comparison to a commercially available system of laminar sheer stress to track live cell calcium influx in response to fluid flow. In addition, we demonstrate the fluid flow-dependent activation of phospho-ERK in these cells, consistent with the findings in other fluid flow devices. This device provides a low cost, multi-functional alternative to currently available systems, while still providing the ability to generate physiologically relevant conditions for studying processes involved in mechanotransduction in vitro.
Subject(s)
Cytophotometry/instrumentation , Mechanotransduction, Cellular , Animals , Calcium Signaling , Cell Culture Techniques/instrumentation , Cells, Cultured , Hydrodynamics , Osteoblasts/physiology , Rats , Single-Cell Analysis/instrumentationSubject(s)
Cytophotometry/methods , Drug Discovery/methods , High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Animals , Cytophotometry/instrumentation , Drug Discovery/instrumentation , High-Throughput Screening Assays/instrumentation , Humans , Mass Spectrometry/instrumentation , Palladium/chemistryABSTRACT
Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. © 2016 International Society for Advancement of Cytometry.
Subject(s)
Cytophotometry/methods , Mass Spectrometry/methods , Osmium Tetroxide/chemistry , Ruthenium Compounds/chemistry , Single-Cell Analysis/methods , Staining and Labeling/methods , Amino Acids/chemistry , Antibodies, Monoclonal/chemistry , Antigens, CD/analysis , Cell Line, Tumor , Chelating Agents/chemistry , Cytophotometry/instrumentation , Fatty Acids/chemistry , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Jurkat Cells , Leukocytes, Mononuclear/classification , Leukocytes, Mononuclear/cytology , Mass Spectrometry/instrumentation , Palladium/chemistry , Single-Cell Analysis/instrumentationABSTRACT
Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of mass (CoM) within a selected region based on the weighted intensity values of structure forming pixels, selected by upper and lower intensity thresholds. Using synthetic and real biological samples, we prove that the displacement of the weighted CoM reliably characterizes the motility of dendritic protrusions. Additionally, guidelines to define optimal parameters of live cell recordings from dendritic protrusions are provided. © 2014 International Society for Advancement of Cytometry.
Subject(s)
Cytophotometry/instrumentation , Dendrites/ultrastructure , Pseudopodia/ultrastructure , Synapses/ultrastructure , Time-Lapse Imaging/instrumentation , Animals , Cell Movement , Cytophotometry/methods , Dendrites/metabolism , Embryo, Mammalian , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/metabolism , Hippocampus/ultrastructure , Image Processing, Computer-Assisted , Mice , Primary Cell Culture , Pseudopodia/metabolism , Synapses/metabolism , Time-Lapse Imaging/methodsABSTRACT
The mechanical properties of cells are known to be a label-free, inherent marker of biological function in health and disease. Wide-spread utilization has so far been impeded by the lack of a convenient measurement technique with sufficient throughput. To address this unmet need, we have recently introduced real-time deformability cytometry (RT-DC) for continuous mechanical single-cell classification of heterogeneous cell populations at rates of several hundred cells per second. Cells are driven through the constriction zone of a microfluidic chip leading to cell deformations due to hydrodynamic stresses only. Our custom-built image processing software performs image acquisition, image analysis and data storage on the fly. The ensuing deformations can be quantified and an analytical model enables the derivation of cell material properties. Performing RT-DC we highlight its potential to identify rare objects in heterogeneous suspensions and to track drug-induced changes in cells. In summary, RT-DC enables marker-free, quantitative phenotyping of heterogeneous cell populations with a throughput comparable to standard flow cytometry.
Subject(s)
Cytophotometry/instrumentation , Cytophotometry/methods , Equipment Design , Flow Cytometry/methods , HL-60 Cells , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Humans , Hydrodynamics , Image Processing, Computer-Assisted , Lab-On-A-Chip DevicesABSTRACT
Bacteria tests are conducted for quality control in many different industries. However, the cultivation method takes a long period of time to obtain results and there are more than a few bacteria that are difficult to cultivate. We have focused on the autofluorescence substance in the bacteria to detect them, and developed a sensor to measure the bacteria in real-time, without any pretreatments or addition of any reagents. This system uses a 405nm laser focused on the sample flowing through the flow-cell in order to detect the fluorescent light from the bacteria as well as scattered light. Fluorescent light and scattered light are separated by a dichroic mirror, and the number of viable particles (bacteria) and that of non-viable particles are obtained. We tested this system using fluorescent polystyrene latex particles and several bacterial strains, and confirmed that it had good detection capability. We believe that this system will become a next-generation bacteria detection system and help the introduction of PAT (process analytical technology) to all areas where real time and on-site detection is needed.
Subject(s)
Bacteria/isolation & purification , Cytophotometry/methods , Fresh Water/microbiology , Bacteria/chemistry , Computer Systems , Cytophotometry/instrumentation , Ecosystem , Equipment Design , FluorescenceABSTRACT
INTRODUCTION: Haematological analysis of body fluids (BF) specimens can provide clinicians with valuable diagnostic information because it can indicate one of several serious medical conditions. Although up to now the microscopic counting and the differentiation of WBC in a BF smear have been used as a reference. The introduction of semiautomated and automated methods of analysis has reduced interoperator variability and improved turnaround time and precision. The aim of our study was to evaluate the accuracy and the correlation between the three methods and with the reference method. METHODS: We examined 110 body fluid samples. Total counting of each sample has been conducted with all systems: Pentra DX120, ADVIA 2120 and XE-2100 and the manual method. RESULTS: We found statistically significant correlation between the data obtained in the ascitic and pleuric liquid but not in the cerebrospinal fluid. CONCLUSION: The introduction of automated method for BF analysis is more and more useful in the routine job of a laboratory analysis. It is therefore very important to evaluate the performance of the different automated haematology technologies, because there is a lack of literature in this field. The comparison between the Pentra DX 120, the other technologies and the manual counting showed instrumental overlapping capabilities.
Subject(s)
Body Fluids/cytology , Cytophotometry/instrumentation , Cytophotometry/methods , Microscopy , Adult , Aged , Aged, 80 and over , Ascitic Fluid/cytology , Ascitic Fluid/pathology , Cerebrospinal Fluid/cytology , Female , Humans , Leukocyte Count/instrumentation , Leukocyte Count/methods , Male , Microscopy/instrumentation , Microscopy/methods , Middle Aged , Reference Values , Young AdultABSTRACT
Cytometry is a key technology for immunology. It allows researchers to scrutinize the cells of the immune system in molecular detail, and to assess phenotype and function at the level of individual cells, no matter how rare these cells may be. The International Society for the Advancement of Cytometry, ISAC, by way of its meetings, online resources and publications (e.g. Cytometry Part A and Current Protocols in Cytometry, which are all published by Wiley) track the ever advancing developments regarding cytometry instrumentation and reagents, and the analysis of complex data sets. In June this year in Leipzig, Germany, ISAC held its annual conference "CYTO 2012", a marketplace of innovation in cytometry.
Subject(s)
Allergy and Immunology/instrumentation , Cytophotometry/instrumentation , Cytophotometry/methods , Animals , Congresses as Topic , Germany , HumansABSTRACT
In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.
Subject(s)
Cytophotometry/methods , Animals , Cell Survival , Cytophotometry/economics , Cytophotometry/instrumentation , HumansABSTRACT
Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.
Subject(s)
Cytophotometry/methods , Escherichia coli/isolation & purification , Single-Cell Analysis/methods , Staining and Labeling/methods , Cell Membrane/chemistry , Cell Separation , Chelating Agents/chemistry , Cytophotometry/instrumentation , Escherichia coli/classification , Lanthanoid Series Elements/chemistry , Lectins/chemistry , Lipopolysaccharides/chemistry , Mass Spectrometry , Mutation , Pentetic Acid/chemistryABSTRACT
We incorporate optics and an ICCD to record the two-dimensional angular optical scattering (TAOS) patterns retrieved from single aerosolized cells. We analyze these patterns by performing autocorrelations and demonstrate that we are able to retrieve cell size from the locations of the secondary maxima. Additional morphological information is contained in the autocorrelation functions and decay rate of the heights of the autocorrelation peaks. We demonstrate these techniques with C6 and Y79 cells, which are readily distinguishable. One key advantage of this methodology is that there is no requirement for antibody and fluorescent labeling molecules.
Subject(s)
Cells/cytology , Cytophotometry/methods , Light , Scattering, Radiation , Animals , Cell Line, Tumor , Cytophotometry/instrumentation , Lasers , Models, Theoretical , RatsABSTRACT
The aim of the present study was to evaluate the effectiveness of Cellscan in identifying culprit drugs causing cutaneous adverse drug reaction. It was a prospective study with 3 months follow up conducted at the Departments of Dermatology, Internal Medicine and Dermatology Outpatient Clinic, Chaim Sheba Medical Center, Tel Hashomer, Israel. The study included 36 patients with cutaneous reaction suspected to be secondary to drugs, treated with a total number of 148 drugs. All patients and drugs were classified to three probability groups according to accepted clinical criteria. The effectiveness of the Cellscan test in identifying the culprit drug was addressed according to the clinical probability for cutaneous drug reaction, the drug class and the type of rash. Data analysis according to the clinical probability for cutaneous drug reaction revealed that patients in the moderate and high probability groups had a high test sensitivity of 77.7% and 83.3% with specificity of 71% and 63%, respectively, in identifying the culprit drug. Classifying the data according to drug classes, revealed that for the antibiotic and cardiovascular drug classes the sensitivity of the test was 100% and 92% with specificity of 83.3% and 55.5%, respectively, in identifying the culprit drug. Finally, the classification of patients according to the type of rash revealed a high evaluating accuracy for culprit drugs in maculopapular rashes with sensitivity and specificity of 90% and 60.4%, respectively. The results of this study imply that the Cellscan test is it a good practical tool for identifying the culprit drug in cutaneous adverse drug reaction.
Subject(s)
Cytophotometry/methods , Drug Eruptions/etiology , Drug-Related Side Effects and Adverse Reactions , Fluorescence Polarization/methods , Adult , Aged , Cytophotometry/instrumentation , Female , Fluorescence Polarization/instrumentation , Follow-Up Studies , Humans , Male , Middle Aged , Pharmaceutical Preparations/analysis , Probability , Prospective Studies , Sensitivity and SpecificityABSTRACT
A smart miniaturized system is being proposed for the isolation and characterization of circulating tumor cells (CTCs) directly from blood. Different microfluidic modules have been designed for cell enrichment and -counting, multiplex mRNA amplification as well as DNA detection. With the different modules at hand, future effort will focus on the integration of the modules in a fully automated, single platform.
Subject(s)
Cell Separation/instrumentation , Lab-On-A-Chip Devices , Neoplasms/blood , Neoplastic Cells, Circulating , Cell Separation/methods , Cytophotometry/instrumentation , Cytophotometry/methods , DNA, Neoplasm/isolation & purification , Humans , Neoplasms/genetics , Neoplasms/pathology , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methodsABSTRACT
Quantitative nuclei DNA content measurement based on Feulgen reaction and the analysis of CCD images was tested. The measurements were performed in monochorome CCD option (650 X 514 pixels) with the wavelength 551 nm. The linear dependence of photomatrix elements signals on the falling light was shown with the use of multigraded light absorption filter. The optimal microscope and camera setting and an approach for elimination of the optic blur were found. We have shown that the proper Feulgen fluorescence does not affect our measurements. Densitometric DNA content measurements of blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens and Rana arvalis) showed good consistence to the literature data (http://www.genomesize.com). The precision of our approach is comparable to the other known methods. Current improvement of CCD technical parameters and wide usage of CCD cameras in biological applications gives perspectives for the suggested approach.