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BioTechniques Apr 2022Flow cytometry is a single-cell technology that measures scatter and fluorescence to establish a set of unique cellular properties. Flow cytometry is used in many areas... (Review)
Review
Flow cytometry is a single-cell technology that measures scatter and fluorescence to establish a set of unique cellular properties. Flow cytometry is used in many areas of science, in particular biotechnology and medicine, but also in industrial applications. Flow cytometry can identify multiple phenotypic subsets from a mixture, select a single cell and even isolate that cell by a process called cell sorting. The field is currently undergoing dramatic changes. We are moving rapidly from the polychromic flow cytometry that has been the go-to technology for 45 years to spectral flow cytometry, which is now the most significant change in nearly half a century of flow cytometry. With change comes opportunity. Even spectral flow cytometry will morph into second-generation spectral flow cytometry within 5 years. New, exciting features will open up molecular diagnostics and physiology to flow cytometry.
Topics: Cell Separation; Flow Cytometry
PubMed: 35369735
DOI: 10.2144/btn-2022-0005 -
International Journal of Molecular... Jul 2015The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging... (Review)
Review
The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.
Topics: Animals; Cell Separation; Humans; Marketing; Patents as Topic; Single-Cell Analysis
PubMed: 26213926
DOI: 10.3390/ijms160816897 -
Current Protocols in Immunology Aug 2015Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative...
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.
Topics: Animals; Ascitic Fluid; Bone Marrow Cells; Cell Separation; Mice; Neutrophils
PubMed: 26237011
DOI: 10.1002/0471142735.im0320s110 -
Analytical Chemistry Jan 2020
Review
Topics: Animals; Cell Movement; Cell Separation; Flow Cytometry; Humans
PubMed: 31808677
DOI: 10.1021/acs.analchem.9b05357 -
Trends in Biotechnology Jun 2021Technologies capable of cell separation based on cell images provide powerful tools enabling cell selection criteria that rely on spatially or temporally varying... (Review)
Review
Technologies capable of cell separation based on cell images provide powerful tools enabling cell selection criteria that rely on spatially or temporally varying properties. Image-based cell sorting (IBCS) systems utilize microfluidic or microarray platforms, each having unique characteristics and applications. The advent of IBCS marks a new paradigm in which cell phenotype and behavior can be explored with high resolution and tied to cellular physiological and omics data, providing a deeper understanding of single-cell physiology and the creation of cell lines with unique properties. Cell sorting guided by high-content image information has far-reaching implications in biomedical research, clinical medicine, and pharmaceutical development.
Topics: Cell Physiological Phenomena; Cell Separation; Microfluidics
PubMed: 33190968
DOI: 10.1016/j.tibtech.2020.10.006 -
Analytical and Bioanalytical Chemistry Aug 2010Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods... (Review)
Review
Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible.
Topics: Animals; Cell Separation; Cells; Humans; Microfluidic Analytical Techniques
PubMed: 20419490
DOI: 10.1007/s00216-010-3721-9 -
Journal of Immunological Methods Nov 2020Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two... (Comparative Study)
Comparative Study
Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two types of cells are the major responders by producing and releasing inflammatory mediators. Isolation of microglia and astrocytes from CNS tissue provides a powerful tool to study basic cell biology and examine the effects of in vivo treatments on microglia and astrocytes immunophenotype and function. The widely used approach of enrichment microglia and astrocytes from CNS was MACS (Magnetic activated cell sorting) and FACS (Fluorescence activated cell sorting). Here we described an optimized protocol of enzymatic dissociation generating single cell suspensions from brain tissue. Then the ability of the two methods to isolate microglia and astrocytes from brain dissociated cells was compared. Both MACS and FACS processing could obtain microglia and astrocytes with high viability (>85%). Microglia sorted by MACS comprises a slight myeloid cells contamination but with a little bit higher efficiency than that sorted by FACS. MACS processing was faster than FACS for either single or multiple samples. ACSA2 can be used to isolate astrocytes from both postnatal and adult brain, and is more suitable for purify astrocytes from newborn. FACS could get purer microglia which is helpful for deep sequencing and other related research. ACSA2 is a good marker of astrocytes.
Topics: Animals; Astrocytes; Biomarkers; Brain; CD11b Antigen; Cell Separation; Cell Survival; Cells, Cultured; Flow Cytometry; Gene Expression Regulation; Immunomagnetic Separation; Leukocyte Common Antigens; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Phenotype
PubMed: 32810482
DOI: 10.1016/j.jim.2020.112834 -
Nature Communications Jul 2020The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade....
The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.
Topics: Animals; Cell Separation; Humans; Spectrum Analysis, Raman
PubMed: 32651381
DOI: 10.1038/s41467-020-17285-3 -
Current Opinion in Chemical Biology Aug 2016Single cell Raman spectra (SCRS) are intrinsic biochemical profiles and 'chemical images' of single cells which can be used to characterise phenotypic changes,... (Review)
Review
Single cell Raman spectra (SCRS) are intrinsic biochemical profiles and 'chemical images' of single cells which can be used to characterise phenotypic changes, physiological states and functions of cells. On the base of SCRS, Raman activated cell sorting (RACS) provides a label-free cell sorting approach, which can link single cells to their chemical or phenotypic profiles. Overcoming naturally weak Raman signals, establishing Raman biomarker as sorting criteria to RACS and improving specific sorting technology are three challenges of developing RACS. Advances on Raman spectroscopy such as stimulated Raman scattering (SRS) and pre-screening helped to increase RACS sorting speed. Entire SCRS can be characterised using pattern recognition methods, and specific Raman bands can be extracted as biomarkers for RACS. Recent advances on cell sorting technologies based on microfluidic device and surface-ejection enable accurate and reliable single cell sorting from complex samples. A high throughput RACS will be achievable in near future by integrating fast Raman detection system such as SRS with microfluidic RACS and Raman activated cell ejection (RACE).
Topics: Biomarkers; Cell Separation; Single-Cell Analysis; Spectrum Analysis, Raman
PubMed: 27100046
DOI: 10.1016/j.cbpa.2016.04.002 -
Cytometry. Part a : the Journal of the... Sep 2022Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants... (Review)
Review
Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.
Topics: Cell Separation; Flow Cytometry; Protoplasts; Suspensions
PubMed: 34028996
DOI: 10.1002/cyto.a.24461