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Cell sorting

Cell sorting is a fundamental laboratory technique in biology and medicine that enables the physical separation of heterogeneous cell populations into distinct subpopulations based on specific physical, biochemical, or fluorescent characteristics.^[1] This process typically integrates flow cytometry for high-throughput analysis with mechanisms for precise isolation, allowing researchers to purify cells of interest from complex mixtures such as blood, tissues, or cell cultures.^[2] Often referred to as fluorescence-activated cell sorting (FACS), it achieves high purity levels exceeding 95% and can process thousands of cells per second, making it indispensable for applications in immunology, stem cell research, and cancer studies.^[1] The origins of cell sorting trace back to 1965, when Mack Fulwyler developed the first device capable of separating biological cells by volume using an inkjet printer-inspired method involving electrical charging of particles in a conducting medium.^[3] This impedance-based system laid the groundwork for modern sorters by demonstrating the feasibility of automated, volume-dependent fractionation.^[4] A pivotal advancement occurred in 1972 with the introduction of fluorescence-activated cell sorting by Bonner, Hulett, Sweet, and Herzenberg, who engineered an instrument that labeled cells with fluorescent dyes, incorporated them into a liquid stream, and deflected charged droplets containing target cells via electrostatic fields.^[5] This innovation, commercialized as the BD FACS system, revolutionized cell isolation by enabling sorting based on multiple fluorescent parameters, far surpassing earlier mechanical or density-gradient methods in speed and specificity.^[1] At its core, cell sorting operates on the principles of hydrodynamic focusing and optical detection: cells are suspended in a sheath fluid, accelerated through a narrow nozzle to form a single-file stream, and interrogated by lasers that measure light scatter (indicating size and granularity) and fluorescence from bound antibodies or dyes.^[2] Sorting decisions are made in real-time by software analyzing these signals against user-defined gates, triggering separation via techniques such as electrostatic deflection in jet-in-air systems (sorting up to 100,000 events per second) or mechanical pinching in microfluidic devices for gentler handling of fragile cells.^[1] Key variants include label-free methods like dielectrophoresis or acoustophoresis, which exploit intrinsic cellular properties without dyes, and image-activated sorting, which uses microscopy for morphological assessment.^[6] Cell sorting's versatility extends to diverse applications, including the isolation of rare immune subsets for vaccine development, enrichment of tumor-infiltrating lymphocytes for immunotherapy, and purification of viable single cells for genomic sequencing or clonal expansion. In regenerative medicine, it facilitates the selection of pluripotent stem cells or progenitors based on surface markers, enhancing differentiation protocols. Ongoing advancements, such as integration with CRISPR editing or AI-driven gating, continue to expand its precision and throughput, addressing challenges like maintaining high cell viability post-sorting and scalability for clinical use.^[7]

Principles and Fundamentals

Definition and Importance

Cell sorting is the process of separating heterogeneous cell populations into distinct subpopulations based on their specific physical, chemical, or biological properties, such as size, density, surface markers, or fluorescence signals.^[8]^[9] This technique enables the isolation of rare or target cells from complex mixtures, providing a foundational tool for advancing biological research and clinical applications. The importance of cell sorting spans multiple fields, including immunology, where it allows the purification of specific immune cell subsets to study responses and develop immunotherapies; oncology, facilitating the separation of tumor cells to investigate heterogeneity and drug resistance; stem cell research, through enrichment of hematopoietic or mesenchymal stem cells for regenerative therapies; and diagnostics, where it aids in identifying pathological cells for disease detection.^[10]^[11]^[12] By achieving high-purity isolation, cell sorting supports downstream analyses like functional assays, genetic sequencing, and protein profiling, enhancing the accuracy of experimental outcomes.^[13] Key benefits include its scalability, accommodating bulk processing of millions of cells or precise isolation at the single-cell level, and its compatibility with omics technologies such as transcriptomics and proteomics, where sorted cells serve as high-quality inputs for comprehensive molecular profiling.^[14]^[15] Since the 1970s, when fluorescence-based sorting emerged, this technology has broadly impacted personalized medicine by enabling tailored cell-based treatments, such as autologous stem cell transplants and targeted cancer immunotherapies.^[16]

Underlying Physical and Biological Mechanisms

Cell sorting relies on fundamental physical principles to manipulate and separate cells within fluidic systems. Hydrodynamic focusing aligns cells into a single-file stream by surrounding the sample with a sheath fluid, creating laminar flow that confines particles to the center of the channel at low Reynolds numbers, enabling precise interrogation and sorting in techniques like flow cytometry.^[17]^[18] This process achieves positioning accuracy within 1 micrometer, with the sample core diameter expanding as flow rates increase from 10 to 120 μL/min, which can influence measurement variability.^[18] Electrostatic deflection, used in droplet-based sorters, charges microdroplets containing target cells via a voltage pulse and directs them through an electric field into collection vessels, allowing simultaneous sorting of up to six populations based on optical properties.^[18] Centrifugal forces, generated in curved microchannels or rotating devices, induce secondary flows like Dean vortices that migrate cells radially according to their size and density, facilitating separation without labels.^[17] These forces scale with the Dean number, defined as De = [Re](/page/Re) \sqrt{[D_h](/page/Hydraulic_diameter) / [R](/page/R)}, where [Re](/page/Re) is the Reynolds number, [D_h](/page/Hydraulic_diameter) is the hydraulic diameter, and [R](/page/R) is the channel curvature radius.^[17] Biologically, cell sorting targets specific molecular features to distinguish subpopulations. Cell surface markers, such as antigens (e.g., CD3 on T cells or CD19 on B cells), serve as primary identifiers, binding selectively to antibodies for detection and isolation.^[19] Intracellular components, including transcription factors like FoxP3 or proliferation markers like Ki67, can be accessed in fixed and permeabilized cells to reveal functional states, though this compromises cell viability.^[19] Viability indicators, such as membrane-exclusion dyes (e.g., propidium iodide), exclude non-viable cells by penetrating compromised membranes, ensuring sorted populations consist of live cells suitable for downstream applications.^[19] Two broad strategies underpin cell sorting: label-free and labeled approaches. Label-free methods exploit intrinsic biophysical properties, separating cells by size through filtration or inertial focusing, density via sedimentation or centrifugation, or deformability using microstructures that deform flexible cells differently from rigid ones.^[20] For instance, density-based separation follows Stokes' law for sedimentation velocity, given by

v = \frac{2r^2 (\rho_p - \rho_f) g}{9 \eta},

where v is the terminal velocity, r is the particle radius, \rho_p and \rho_f are the particle and fluid densities, g is gravitational acceleration, and \eta is the fluid viscosity; denser cells sediment faster under enhanced gravity in centrifugation.^[21] Labeled sorting, in contrast, uses affinity binding where antibodies or ligands specifically attach to surface markers, enabling targeted capture via fluorescence or magnetism, though it requires prior knowledge of the markers.^[20] These mechanisms collectively enable high-purity isolation, with label-free techniques achieving resolutions down to 5 μm in size and deformability differences of about 7%.^[20]

Historical Development

Early Techniques and Milestones

The earliest approaches to cell sorting relied on manual micromanipulation, a labor-intensive technique developed in the early 20th century that involved using fine glass micropipettes under an inverted microscope to physically isolate individual cells from a heterogeneous suspension.^[22] This method, often employed in hematology for selecting rare cell types such as leukocytes, allowed for precise single-cell picking but was limited to low throughput, typically processing only a few dozen cells per hour due to its dependence on skilled operators.^[23] Parallel to micromanipulation, density gradient centrifugation emerged as a foundational pre-1970s technique for bulk cell separation, exploiting differences in cell buoyancy to fractionate populations based on physical properties. Early implementations in the 1930s used nonaqueous gradients for particle separation, but by the 1950s, aqueous methods like bovine serum albumin gradients were adapted for isolating blood cells in hematological studies.^[24] A key advancement came in the 1960s with the Ficoll-Hypaque method, introduced by Bøyum in 1968, which combined the polysaccharide Ficoll with the dense contrast agent Hypaque to create a stable gradient for efficient isolation of mononuclear cells from peripheral blood via a single centrifugation step.^[25] This technique improved upon prior gradients by reducing osmotic stress on cells and enabling higher purity separations, though it lacked specificity for subpopulations beyond broad density classes like lymphocytes versus granulocytes.^[25] Significant milestones in the 1950s and 1960s laid the groundwork for automated flow-based sorting. In 1953, Wallace H. Coulter invented the impedance-based particle counter, patented as a method to measure cell volume by detecting changes in electrical resistance as cells passed through an aperture, revolutionizing hematological cell enumeration and providing the core principle for flow cytometry.^[26] Building on this, Mack J. Fulwyler pioneered the first electrostatic cell sorter in 1965 at Los Alamos National Laboratory, integrating Coulter volume sensing with inkjet printer technology to charge and deflect droplets containing selected cells, as detailed in his seminal Science publication and subsequent 1968 patent.^[3]^[27] These innovations enabled the first automated sorting of biological cells by size, with early applications in hematology for purifying cell fractions to study blood disorders and immune responses.^[28] Despite these advances, early cell sorting methods suffered from inherent limitations that constrained their utility. Manual micromanipulation and density gradients offered poor specificity, relying solely on morphological or density differences without molecular markers, often resulting in contaminated fractions unsuitable for downstream analyses.^[29] Electrostatic sorters like Fulwyler's prototype achieved only modest throughputs of around 100-300 cells per second and were prone to clogging or imprecise deflection, limiting scalability for clinical or large-scale research in hematology.^[30] Overall, these techniques prioritized conceptual proof-of-principle over high-efficiency separation, setting the stage for later refinements.^[31]

Evolution to Modern Systems

The development of fluorescence-activated cell sorting (FACS) in the 1970s marked a pivotal breakthrough in cell sorting technology, enabling the automated separation of cells based on fluorescent labeling. In 1972, Leonard Herzenberg and colleagues at Stanford University introduced the first FACS instrument, which utilized flow cytometry principles to detect and sort viable cells at high speeds by incorporating them into a liquid stream and deflecting droplets electrostatically.^[32] This innovation, building on earlier flow cytometry concepts, allowed for precise isolation of cell subpopulations, such as those expressing specific antigens, transforming immunological research.^[33] During the 1980s and 1990s, cell sorting advanced through commercialization and diversification of methods, enhancing accessibility and versatility. Becton Dickinson licensed the FACS technology from Stanford and released the first commercial instrument, the FACS II, in 1974, followed by iterative improvements that integrated multi-laser systems and expanded parameter analysis.^[16] Concurrently, magnetic bead-based separation emerged with the invention of Dynabeads by John Ugelstad in 1976, superparamagnetic polystyrene microspheres that enabled gentle, antibody-mediated cell isolation without fluidics, widely adopted for positive and negative selection in research and clinical settings. The late 1990s also saw the rise of microfluidics in cell sorting, with initial demonstrations of microscale devices for dielectrophoretic and hydrodynamic separation, offering miniaturization and reduced sample volumes compared to macroscale systems.^[34] From the 2000s onward, cell sorting evolved toward automation, high-throughput processing, and single-cell precision, fueled by the genomics revolution that demanded resolution of cellular heterogeneity. Integration of robotics and software automation in FACS and microfluidic platforms enabled sorting rates exceeding 10,000 cells per second, while advancements like droplet-based microfluidics facilitated index sorting for downstream genomic analysis.^[35] The 2008 Nobel Prize in Chemistry, awarded to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for the discovery and development of green fluorescent protein (GFP), profoundly impacted flow cytometry by providing brighter, genetically encodable labels that enhanced multiplexing and viability in sorting applications. This period shifted the field from bulk population analysis to precision isolation of individual cells, driven by the post-Human Genome Project boom in transcriptomics and epigenomics, enabling studies of rare events like tumor heterogeneity.^[36]

Primary Sorting Methods

Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting (FACS) is a specialized flow cytometry technique that enables the high-speed separation of heterogeneous cell populations based on their optical properties, particularly fluorescence and light scatter, allowing for the isolation of specific cell subsets with high purity. Developed in the late 1960s, FACS integrates fluidics, optics, and electronics to analyze individual cells in a stream and physically sort them into distinct fractions using electrostatic forces. This method is widely used in immunology, stem cell research, and clinical diagnostics due to its ability to handle multiple parameters simultaneously.^[37]^[19] The core components of a FACS system include the fluidics system, optics for excitation, and detectors for signal collection. The fluidics system employs a sheath fluid, typically a buffered saline solution under pressure, to create hydrodynamic focusing, which aligns cells in a single-file laminar flow through a narrow interrogation point. Optics consist of one or more lasers (e.g., 488 nm argon-ion or 405 nm violet) that excite fluorescent labels on cells, inducing emission and scatter. Detectors, such as photomultiplier tubes (PMTs) or avalanche photodiodes, capture forward scatter (FSC) for cell size, side scatter (SSC) for granularity, and fluorescence signals across multiple wavelengths using dichroic mirrors and bandpass filters.^[19]^[38] The FACS procedure begins with cell labeling using fluorescent antibodies or dyes that bind to surface markers, intracellular proteins, or viability indicators, followed by resuspension in sheath fluid. Cells are then injected into the fluidics system, where hydrodynamic focusing confines them to the center of the stream for precise laser interrogation. As the stream exits the nozzle, a piezoelectric transducer induces oscillations to generate uniform droplets at rates of 10,000–100,000 per second, with each droplet potentially containing a single cell based on timing from the detection point. If a droplet meets predefined criteria (e.g., fluorescence threshold), it is charged by an electrode; uncharged droplets proceed to waste, while charged ones are deflected by high-voltage plates (up to 5,000–10,000 V) into collection tubes or multi-well plates.^[37]^[1] Common fluorescent dyes in FACS include fluorescein isothiocyanate (FITC), which emits green light (excitation ~488 nm, emission ~525 nm) and is frequently conjugated to antibodies for surface marker detection; phycoerythrin (PE), a protein-phycobiliprotein with high quantum yield and orange-red emission (excitation ~488–565 nm, emission ~575–580 nm) for brighter signals; and propidium iodide (PI), a red-emitting DNA-intercalating viability dye (excitation ~535 nm, emission ~617 nm) that excludes live cells by penetrating only compromised membranes. Multiplexing allows simultaneous detection of 10–30 parameters by combining dyes with distinct spectra, such as tandem conjugates (e.g., PE-Cy5) or polymer-based dyes (e.g., Brilliant Violet series), enabling complex phenotyping without spectral overlap.^[19] FACS systems achieve high throughput, with sorting rates typically up to 5,000–10,000 cells per second for high-purity separations (>95%), depending on nozzle size, event rate, and target population frequency.^[39] Signal detection relies on fluorescence intensity, governed by the equation

I = \phi \cdot I_0 \cdot (1 - 10^{-\epsilon c l})

where I is the emitted fluorescence intensity, \phi is the quantum yield, I_0 is the excitation intensity, \epsilon is the molar absorptivity, c is the fluorophore concentration, and l is the optical path length, reflecting the Beer-Lambert absorption principle adapted for emission.^[40]^[41]

Immunomagnetic Cell Sorting

Immunomagnetic cell sorting is a technique that employs superparamagnetic beads coated with specific antibodies to isolate target cells based on surface antigens, leveraging magnetic fields for separation without the need for complex fluidic systems.^[42] The process relies on the beads' superparamagnetic properties, which allow them to be magnetized only in the presence of an external field, enabling reversible retention and minimizing aggregation.^[43] This method is particularly suited for bulk isolation of cell populations, offering a straightforward alternative to flow-based sorting for applications requiring high yields of viable cells. The seminal system, Magnetic-Activated Cell Sorting (MACS), was developed by Miltenyi Biotec and first described in 1990, revolutionizing affinity-based cell enrichment through high-gradient magnetic separation.^[43] MACS operates in positive selection mode, where antibody-bound target cells are retained on a column within a magnetic field, or negative selection mode, where undesired cells are labeled and depleted, leaving the target population in the flow-through.^[44] These modes provide flexibility for enriching rare or abundant cell types, such as immune subsets expressing CD markers, with reported purities exceeding 95% in optimized protocols.^[45] Central to the technique are the superparamagnetic beads, typically 50 nm in diameter and composed of an iron oxide core encapsulated in a biocompatible dextran shell to prevent non-specific binding.^[46] These MACS MicroBeads are covalently conjugated to monoclonal antibodies targeting specific surface antigens, such as CD4 or CD8 for T-cell isolation, ensuring high-affinity binding with minimal epitope masking due to the nano-scale size.^[47] The small bead dimensions allow for efficient cell labeling without altering cellular function, and their superparamagnetic nature—derived from magnetite (Fe₃O₄) nanoparticles—facilitates rapid response to magnetic gradients up to 0.5 Tesla in standard setups.^[45] The procedure begins with incubating the cell suspension with the antibody-bead conjugate for 15-30 minutes at room temperature, promoting specific binding to target antigens.^[42] The mixture is then loaded onto a MACS column, a matrix of ferromagnetic spheres that amplifies the external magnetic field to retain labeled cells while permitting unlabeled cells to elute. After washing to remove unbound material, the column is removed from the magnet, and target cells are gently eluted using buffer, yielding highly viable populations suitable for downstream applications.^[43] Depletion strategies, often via negative selection, can further enhance purity by iteratively removing multiple unwanted subsets in multi-step protocols.^[48] Key advantages include the absence of high-pressure fluidics, reducing shear stress on delicate cells like neurons or stem cells, and exceptional specificity for antigen-mediated isolation.^[44] Automated variants, such as the MultiMACS system, achieve throughputs of up to 10⁹–10¹⁰ cells per hour, making it ideal for clinical-scale processing in immunotherapy.^[49] Overall, this method's simplicity and scalability have established it as a cornerstone for bulk cell purification in research and biomanufacturing.^[45]

Microfluidic Cell Sorting

Microfluidic cell sorting involves lab-on-a-chip devices that integrate microscale channels, typically with dimensions of 10-100 μm, to manipulate and separate cells with high precision. These systems employ pumps, such as syringe or pressure-driven mechanisms, to control laminar flow rates, ensuring predictable cell trajectories without turbulence. Integrated sensors, including optical detectors for fluorescence or impedance-based electrical sensing, enable real-time monitoring and sorting decisions. This setup allows for the processing of small sample volumes, often in the nanoliter range, minimizing reagent use and enabling analysis of rare cell populations.^[34] Active microfluidic sorting methods utilize external fields to direct cells selectively. Dielectrophoresis (DEP) exploits non-uniform alternating current electric fields to induce forces on cells based on their dielectric properties, such as polarizability, enabling label-free or marker-enhanced separation. The DEP force is given by the equation

F_{\text{DEP}} = 2\pi r^3 \epsilon_m \operatorname{Re}[K(\omega)] \nabla E^2,

where r is the cell radius, \epsilon_m is the permittivity of the medium, \operatorname{Re}[K(\omega)] is the real part of the Clausius-Mossotti factor, and E is the electric field strength; cells with positive DEP migrate to high-field regions, while those with negative DEP move away, facilitating continuous-flow sorting at throughputs up to 10,000 cells per second. A seminal demonstration of DEP for marker-specific rare cell enrichment achieved over 200-fold purification of labeled E. coli in a single pass. Acoustic sorting, or acoustophoresis, uses ultrasonic standing or traveling waves to generate acoustic radiation forces that displace cells based on size, compressibility, and density, often without labels. For instance, bulk standing wave acoustophoresis has separated viable from nonviable mammalian cells, such as MCF-7 breast tumor cells, at densities of 10^6 cells/mL and flow rates up to 12 mL/h, preserving cell viability due to its non-contact nature.^[50]^[51]^[34] Passive methods rely on hydrodynamic effects within the channel geometry to achieve separation without external energy inputs beyond flow. Deterministic lateral displacement (DLD) employs an array of microposts spaced to create asymmetric flow bifurcations, where particles larger than a critical diameter (typically 1-10 μm) follow a displacing mode for size-based sieving, while smaller ones remain in the original streamline. This technique, introduced for continuous particle separation, has been applied to isolate blood cells or exosomes with resolutions down to 20% of particle size. Pinched-flow fractionation (PFF) pinches cells into a narrow segment of the channel to align them near one wall, then expands the flow to allow differential migration based on size or deformability; larger cells follow outer streamlines for diversion into collection outlets. Pioneered for micrometer-sized particles, PFF enables high-resolution sorting of deformable cells like leukocytes from erythrocytes.^[52]^[53]^[34] These microfluidic approaches offer distinct advantages, including low sample volumes on the order of nanoliters, which reduce costs and biohazards, and superior resolution for subtle biophysical differences compared to macroscale methods. For example, Dean flow in curved or spiral channels generates secondary vortices that enhance inertial migration, focusing larger cells to outer walls for size-based separation at high throughputs exceeding 10^6 cells per minute, as demonstrated in devices for circulating tumor cell isolation. Overall, microfluidic sorting integrates multiple principles for precise, scalable cell handling in research and clinical settings.^[54]^[34]

Centrifugation-Based Methods

Centrifugation-based methods represent simple, label-free approaches for initial cell enrichment, relying on differences in cell density or sedimentation velocity to separate populations without the need for antibodies or fluorescent markers. These techniques are particularly useful for bulk processing of heterogeneous samples like blood or tissue digests, where rapid isolation of mononuclear cells or other subpopulations is required prior to more specific sorting. By exploiting physical properties under centrifugal force, they achieve high throughput while maintaining cell viability, though they offer limited resolution compared to affinity-based methods.^[55] Isopycnic centrifugation using density gradients, such as those formed with Percoll or Ficoll, separates cells by their buoyant density, where particles migrate until they reach an equilibrium position at the interface matching their density. Percoll, a colloidal silica suspension, allows for customizable gradients with densities up to approximately 1.13 g/mL when adjusted with physiological salts, making it suitable for fragile cells like hepatocytes or enveloped viruses.^[56] In a typical step-gradient protocol, a "100% Percoll" stock (prediluted in isotonic solution) is layered in discrete density steps within a centrifuge tube, the sample is overlaid on the top layer, and centrifugation at 400–800 × g for 20–40 minutes at room temperature equilibrates cells into bands at their isopycnic points; the desired fraction is then collected by aspiration.^[56] Ficoll, a neutral polysaccharide often formulated as Ficoll-Paque with a density of about 1.077 g/mL, operates similarly for isolating peripheral blood mononuclear cells (PBMCs), including lymphocytes and monocytes, by layering diluted whole blood over the gradient and centrifuging without brakes to prevent mixing; PBMCs form a distinct buffy coat at the plasma-Ficoll interface due to their lower density relative to granulocytes and erythrocytes.^[18] These methods yield purities of 90–95% for viable mononuclear cells from blood samples, with recoveries typically exceeding 80%, though they may co-isolate some contaminating subpopulations based on overlapping densities.^[57] Velocity sedimentation, or rate-zonal centrifugation, complements isopycnic methods by separating cells primarily by size and sedimentation speed rather than equilibrium density, using shallow preformed gradients to maintain zone integrity during migration. Cells are loaded as a thin band on top of a gradient medium like sucrose or Ficoll, and upon centrifugation at moderate speeds (e.g., 1,000–2,000 × g), larger or denser cells sediment faster through the gradient, forming discrete zones based on their velocity; fractions are collected by puncturing the tube bottom or unloading.^[58] This technique exploits differences in sedimentation rates for subpopulations with similar densities but varying sizes, such as separating myeloid cells from lymphocytes in bone marrow aspirates.^[59] The sedimentation velocity v relates to the centrifugal field via the sedimentation coefficient s, defined as

s = \frac{v}{\omega^2 r}

where s is expressed in Svedberg units (1 S = 10^{-13} s), \omega is the angular velocity in radians per second, and r is the radial distance from the rotation axis in centimeters; this coefficient inherently accounts for particle size, shape, and density under standardized conditions.^[60] Modern variants like centrifugal elutriation extend these principles to continuous-flow sorting, enabling label-free separation in a specialized rotor where cells enter a spinning chamber and are eluted by balancing centrifugal sedimentation against an opposing buffer flow. Smaller cells elute first at lower flow rates, while larger ones require increased flow or speed to counter their faster sedimentation, allowing sequential collection of size-fractionated populations without halting the process.^[61] This method processes up to 10^8 cells per run with high throughput (e.g., synchronizing 2 × 10^7 G1-phase cells from 1.5 × 10^8 asynchronous eukaryotic cells in under 2 hours at flow rates of 30–60 mL/min), achieving purities of 70–90% for mononuclear fractions while preserving cell cycle integrity and viability.^[61] Elutriation's gentle, non-perturbing nature makes it ideal for downstream functional studies, though it requires dedicated equipment for optimal resolution.^[62]

Advanced and Specialized Techniques

Single-Cell Sorting Approaches

Single-cell sorting approaches focus on techniques that isolate individual cells with high precision, enabling downstream analyses such as clonal expansion or genomic profiling. These methods adapt established sorting principles, such as fluorescence-activated cell sorting (FACS), to deposit single cells into multi-well plates or encapsulate them in microdroplets, ensuring one-to-one correspondence between sorted cells and their phenotypic data.^[63] In FACS-based adaptations, index sorting records the fluorescence parameters of each sorted cell and maps them to specific wells in 96- or 384-well plates, allowing retrospective correlation of surface marker expression with single-cell transcriptomics. This plate deposition technique achieves single-cell isolation by directing charged droplets containing one cell into designated wells, with efficiencies exceeding 95% for viable deposition. Droplet encapsulation variants integrate FACS with microfluidic merging, where sorted cells are combined with lysis buffers in emulsion droplets for immediate RNA capture, facilitating high-throughput single-cell readout without physical plate handling.^[63]^[64]^[65] Microfluidic single-cell sorting employs droplet digital methods, such as generating oil-in-water emulsions to encapsulate individual cells at Poisson-limited distributions, typically achieving 10-30% single occupancy while minimizing empty or multi-cell droplets. These oil emulsions enable sorting based on fluorescence or image analysis, with dielectric or hydrodynamic forces deflecting target droplets for collection. Complementary microwell arrays trap single cells in nanoliter-scale wells for clonal expansion, often using gravity sedimentation or deterministic barcoding to ensure isolation, supporting long-term culture and retrieval for functional assays.^[66]^[67]^[68] Robotic systems automate single-cell deposition by using vision-guided pipetting or acoustic dispensing to place cells into 96- or 384-well plates, with onboard imaging confirming occupancy and viability post-deposition. These platforms, such as impedance-based dispensers, reduce manual intervention and achieve deposition rates of up to 1,000 cells per hour while maintaining spatial resolution for downstream imaging.^[69]^[70] These approaches routinely deliver purities greater than 99% for viable single cells, with post-sort viabilities often above 90% when using gentle buffers and low-pressure sorting. Integration with single-cell RNA sequencing (scRNA-seq) workflows began around 2013, enabling combined phenotypic and transcriptomic profiling through index-sorted libraries.^[71]^[72]^[73] Key challenges in single-cell sorting include preventing channel clogging in microfluidic devices, addressed by inertial focusing or anti-fouling coatings to maintain flow rates above 1,000 cells per second. Encapsulation efficiency is optimized through neutral buoyancy suspensions or dual-emulsion designs, boosting single-cell occupancy to over 50% while reducing shear stress on delicate samples.^[72]^[74]^[75]

Antibody-Independent Methods

Antibody-independent methods for cell sorting exploit intrinsic physical and biochemical properties of cells, such as size, deformability, molecular composition, and morphology, without relying on exogenous labels or affinity agents. These approaches enable the separation of heterogeneous cell populations in a non-invasive manner, preserving cellular viability and native states for downstream analyses. Common techniques include filtration-based sieving, spectroscopic identification, and imaging-based morphological assessment, often integrated into microfluidic platforms for high-throughput processing. Filtration and sieving utilize porous membranes to separate cells primarily by size and deformability, where larger or less deformable cells are retained while smaller ones pass through. Microsieve membranes, fabricated from materials like silicon or silicon nitride, feature high-density arrays of uniform pores typically ranging from 5 to 10 μm in diameter, allowing efficient capture of circulating tumor cells (CTCs) from whole blood samples. For instance, devices with 8 μm pores have demonstrated recovery rates exceeding 82% for tumor cells spiked into blood, with minimal white blood cell contamination due to the deformability differences between CTCs and leukocytes. The efficiency of such filtration can be modeled using an adaptation of Darcy's law for flow through porous media:

Q = \frac{k A \Delta P}{\mu L}

where Q is the volumetric flow rate, k is the membrane permeability, A is the effective filtration area, \Delta P is the pressure drop across the membrane, \mu is the fluid viscosity, and L is the membrane thickness. This equation highlights how optimizing pressure and permeability enhances throughput while maintaining separation selectivity in microfiltration systems. Raman spectroscopy sorting provides label-free identification by detecting inelastic light scattering from molecular vibrations, generating a biochemical fingerprint unique to cell types without perturbing their structure. Integrated systems, such as dielectrophoresis (DEP)-Raman platforms developed since the 2010s, combine Raman imaging with electrical forces to sort cells in real-time based on spectral signatures, achieving throughputs of up to 100 cells per second. For example, DEP-based Raman-activated droplet sorting has enabled the isolation of single cells with over 90% purity by targeting specific vibrational modes associated with lipids or proteins. Recent advancements include stimulated Raman-activated cell ejection (S-RACE), which achieves sorting at ~12-50 events per second with >95% purity and yield, enhancing label-free capabilities as of 2024.^[76] Morphological sorting employs holographic imaging to assess cell shape, volume, and refractive index in flowing suspensions, distinguishing subpopulations based on optical phase shifts without labels. Digital holographic microscopy captures interferometric patterns to reconstruct three-dimensional cell profiles, allowing classification of cells by parameters like dry mass or granularity at rates exceeding 100 cells per second. Such systems have been used to differentiate cancer cells from healthy ones via refractive index variations, with classification accuracies above 95% in microfluidic flow cytometry setups. These methods offer key advantages by avoiding the perturbations from labeling agents, such as altered cellular function or toxicity, which is particularly beneficial for sensitive applications. In microbiome research, Raman-based sorting facilitates the isolation of unculturable bacteria based on metabolic profiles, enabling functional metagenomic studies without genetic modification. For rare event detection, like CTC enumeration in cancer diagnostics, filtration microsieves enhance sensitivity by concentrating low-abundance targets from large blood volumes, supporting viable cell recovery for genomic analysis.

Applications and Challenges

Key Applications in Research and Medicine

In research, cell sorting has been instrumental in isolating specific immune cell subsets, such as regulatory T cells (Tregs) defined by CD4+CD25+ markers, enabling detailed functional analyses of their suppressive roles in autoimmune diseases and transplantation tolerance.^[77] For instance, fluorescence-activated cell sorting (FACS) of CD4+CD25+ Tregs from peripheral blood has achieved purities exceeding 90%, facilitating downstream assays like suppression tests that reveal their therapeutic potential in modulating immune responses.^[78] Similarly, sorting CD34+ hematopoietic stem cells from umbilical cord blood or bone marrow has supported studies on stem cell hierarchy and engraftment, with isolation yields up to 77% and purities around 96%, accelerating research into blood disorders and regenerative therapies.^[79] These high-purity isolations enable 10-fold faster downstream genomic and proteomic analyses compared to unsorted populations, enhancing insights into cellular differentiation pathways.^[6] In oncology research, cell sorting dissects tumor heterogeneity by isolating subpopulations based on surface markers, such as CD44+ cancer stem cells from circulating tumor cells (CTCs), which has elucidated mechanisms of metastasis and drug resistance in breast and other cancers.^[80] This approach has been pivotal in validating CRISPR-based genetic screens, where FACS sorting of edited cell populations by fluorescence reporters identifies genes regulating phenotypes like ferroptosis or immune evasion, with sorting-based screens achieving enrichment factors that pinpoint key regulators in primary immune cells.^[81] During the COVID-19 pandemic, cell sorting profiled immune responses by isolating subsets like activated T cells and myeloid cells from patient blood, revealing distinct immunotypes associated with disease severity and guiding vaccine development through high-dimensional flow cytometry data.^[82] Medically, cell sorting underpins CAR-T cell therapy production by purifying engineered CD19-targeted T cells from patient apheresis products, ensuring high viability and specificity that contribute to clinical remission rates in B-cell malignancies.^[83] In transplantation, magnetic-activated cell sorting of CD34+ cells purifies grafts for hematopoietic stem cell transplants, reducing tumor contamination and improving engraftment outcomes in leukemia patients.^[84] For diagnostics, EpCAM-based sorting enriches CTCs from blood, providing a non-invasive biomarker for monitoring cancer progression, with detection sensitivities that correlate with prognosis in epithelial tumors like breast and ovarian cancer.^[85] Emerging applications include generating organoids from sorted progenitors, such as SOX2+HOPX+ neural cells for brain organoids or CDX2+ intestinal progenitors for gut models, which replicate tissue architecture for drug screening and disease modeling.^[86] Integration with spatial transcriptomics involves sorting cells prior to sequencing, enhancing resolution in mapping cellular neighborhoods in tissues like the kidney, where co-embedding of sorted single-cell data with spatial profiles elucidates mouse-to-human translational insights.^[87] These advancements leverage sorting purities above 90% to streamline workflows, enabling rapid validation of therapeutic targets in precision medicine.^[88]

Limitations and Future Directions

Despite their utility, cell sorting techniques face several limitations that can impact their reliability and applicability. High-pressure fluidics and shear forces in methods like fluorescence-activated cell sorting (FACS) often induce sorter-induced cell stress (SICS), leading to reduced viability, particularly for delicate or primary cells, with post-sorting viability frequently falling below 80% in sensitive populations due to decompression shock and mechanical damage.^[89]^[90] Additionally, the high cost of instruments, exceeding $100,000 for standard flow cytometers and reaching up to $500,000 or more for advanced sorters, limits accessibility for many laboratories.^[91] Scalability poses another challenge, especially for isolating rare cell populations comprising less than 0.1% of samples, as it requires processing large volumes to achieve sufficient yields, often compromising purity or efficiency.^[1] Contamination risks further complicate workflows; antibody cross-reactivity can result in non-specific binding and mis-sorting of off-target cells, while aerosol generation in FACS systems raises biosafety concerns by potentially dispersing viable pathogens or fluorophores.^[92]^[93] Looking ahead, innovations are addressing these drawbacks through AI-driven approaches, such as real-time image analysis for label-free sorting, which have gained traction since the early 2020s to enhance accuracy and reduce stress by enabling morphology-based decisions without fluorescent labels.^[94] In 2025, advancements include AI systems for label-free sorting in stem cell therapy and analysis of drug-resistant populations using deep learning on cell morphology.^[95]^[96] Portable devices, like microfluidic-based sorters, are emerging to lower costs and improve field applicability, offering gentle handling for point-of-care use.^[97] Hybrid systems combining FACS with microfluidics promise to mitigate shear stress while maintaining high throughput, integrating electrostatic deflection with chip-based flow for more robust isolation.^[98] As of 2025, key trends include in-line integration of cell sorting with CRISPR editing, allowing immediate enrichment of edited cells post-transfection to boost efficiency in gene therapy workflows, and the adoption of quantum dot labels for superior multiplexing, enabling simultaneous detection of 20+ markers with minimal spectral overlap to overcome traditional fluorophore limitations.^[99]^[100] Potential advancements in in vivo sorting, leveraging targeted probes for non-invasive cell isolation directly in tissues, could expand applications beyond ex vivo processing, though challenges in biocompatibility remain. Projections indicate photonics-enabled systems could achieve throughputs exceeding 1 million cells per second by optimizing optical detection and parallel processing, dramatically scaling analysis for clinical-scale therapies.^[101]

References

[1]
Flow cytometry and cell sorting - PMC - PubMed Central - NIH
Cell sorters use one of several mechanisms to physically separate cells into subpopulations based on the results of cytometric analysis (2). While other ...
[2]
Flow cytometry and cell sorting - Frontiers
Cell sorting (sometimes referred to as fluorescence-activated cell sorting or FACS) is an important extension of flow cytometry (1). Non-sorting flow ...<|separator|>
[3]
Electronic Separation of Biological Cells by Volume - Science
A device capable of separating biological cells (suspended in a conducting medium) according to volume has been developed.
[4]
Electronic separation of biological cells by volume - PubMed
A device capable of separating biological cells (suspended in a conducting medium) according to volume has been developed.
[5]
Fluorescence Activated Cell Sorting - AIP Publishing
Mar 1, 1972 · An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream.
[6]
Cell Separations and Sorting - PMC - PubMed Central - NIH
This review will cover methods for cell separation and sorting based on numerous phenomena, such as acoustophoresis, affinity, dielectrophoresis and inertial ...
[7]
Fluorescence-activated cell sorting: An overview - Abcam
Fluorescence-activated cell sorting (FACS) is one of the most effective, widely used, laser-based methods for isolating and analyzing individual cells.Facs Workflow: From Sample... · Facs Reagents And Antibodies · Applications Of Facs
[8]
Fluorescence activated cell sorting - PubMed
Fluorescence activated cell sorting. Rev Sci Instrum. 1972 Mar;43(3):404-9. doi: 10.1063/1.1685647. Authors. W A Bonner, H R Hulett, R G Sweet, L A Herzenberg.
[9]
Progress of Cell Sorting in Flow Cytometry - Cai - Wiley Online Library
Mar 19, 2025 · This paper reviews the principles and current development of conventional and microfluidic sorting approaches in flow cytometry2 Detection Principle Of... · 2.2 Optical Detection · 4 Applications Of Flow Cell...<|control11|><|separator|>
[10]
Cell Sorting: Techniques, Applications and Future Directions
Cell sorting is a vital process in biological research involving isolating a single cell or a homogeneous population of cells from a complex mixture.Cell Sorting Techniques · Positive/negative Selection · Label-Free MethodsMissing: developmental | Show results with:developmental
[11]
Cell Separation and Cell Isolation: Techniques, Methods, Applications
Cell separation, also commonly referred to as cell isolation or cell sorting, is a process to isolate one or more specific cell populations from a heterogeneous ...
[12]
https://orip.nih.gov/about-orip/research-highlights/going-flow-biological-cell-sorting
[13]
Cell Sorting and Its Significance in Stem Cell Research
Nov 16, 2017 · Cell sorting allows the separation of cells based on their intra- or extracellular properties, including DNA, RNA, and protein interactions, ...
[14]
“Going with the Flow”—Biological Cell Sorting - NIH ORIP
Oct 31, 2017 · Cell sorting and the downstream analysis can assist in the diagnosis of a variety of disorders and provide data regarding cellular mechanisms ...Missing: oncology | Show results with:oncology
[15]
FACS Cell Sorting: A Method for Separating Cell Populations
Its ability to perform multiparameter, marker-based sorting at single-cell resolution ensures high purity and viability critical for sensitive downstream assays ...Where Facs Cell Sorting Is... · How Facs Cell Sorting Works... · Benefits Of Facs Cell...
[16]
Large Particle Fluorescence-Activated Cell Sorting Enables High ...
Aug 15, 2019 · Large Particle Fluorescence-Activated Cell Sorting Enables High-Quality Single-Cell RNA Sequencing and Functional Analysis of Adult ...Missing: purity | Show results with:purity
[17]
Single Cell Multi-Omics Technology: Methodology and Application
The standard protocol is to get viable, intact cells by mechanical or enzymatic dissociation and then capture single cells from the dissociated cell suspension.
[18]
In Search of FACS: The History of Fluorescence Activated Cell Sorting
Sep 12, 2024 · Aside from new labels and increased sorting capacity, FACS functions the same today as it did in the 1970s when it was commercialized. However, ...Merging Fluorescence With... · Facs Goes Commercial · Cell Sorting Gets An Upgrade
[19]
Hydrodynamics in Cell Studies | Chemical Reviews - ACS Publications
Feb 8, 2018 · For nonadherent cells, hydrodynamic cell manipulation techniques for sorting, counting, focusing and trapping cells are discussed in detail. In ...
[20]
Guidelines for the use of flow cytometry and cell sorting in ...
This is an overview and a suggestion of procedures, which should help to get the best results out of your flow data.
[21]
Flow Cytometry: An Overview - PMC - NIH
Following protein transport inhibitor treatment, cells are stained for viability markers and cell surface markers, then fixed and permeabilized for ...
[22]
Label-free cell separation and sorting in microfluidic systems - NIH
This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations.
[23]
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/micromanipulation
[24]
Cell isolation and separation: Essential techniques - Abcam
Cell isolation separates individual cells, while cell separation isolates specific types based on size, shape, surface markers, or density.
[25]
Micromanipulation - an overview | ScienceDirect Topics
Micromanipulation is a technical method in which a micromanipulator is used to separate cells or early embryos under a high-power inverted microscope.
[26]
Density Gradient - an overview | ScienceDirect Topics
Behrens (1938) used nonaqueous gradients in the earliest report on the use of density gradient centrifugation for the separation of particles. About the ...
[27]
https://patents.google.com/patent/US3380584A/en
[28]
Flow Cytometry History - News-Medical
Flow cytometry based on impedance and using the Coulter principle was first made available to Wallace H. Coulter in the U.S. under the patent number 2656508 ...
[29]
Particle separator - US3380584A - Google Patents
Mack J. F u/wy/er April 30, 1968 M. .1. FULWYLER PARTICLE SEPARATOR 5 Sheets-Sheet 2 Filed June 4, 1965 PHOTO-CELL ASSEMBLY Fig. INVENTOR. Mack J. Fu/wy/er ...
[30]
Mack Jett Fulwyler, pioneer of flow cytometry and flow sorting (1936 ...
Sep 14, 2005 · Mack Fulwyler was awarded more than 15 patents for his inventions ... Mack invented droplet cell sorting and helped foster the field of ...
[31]
https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.22296
[32]
High‐Speed Cell Sorting - Leary - 1997 - Current Protocols
May 15, 2001 · First, cell sorters were inherently slower than other cell-separation methods and it was difficult or impossible to isolate enough cells for ...Missing: pre- | Show results with:pre-
[33]
Wallace H. Coulter: Decades of invention and discovery
Apr 17, 2013 · He used the flow cytometry inventions and innovations to drive the hematology instruments from which the greater part of his business success ...
[34]
The History and Future of the Fluorescence Activated Cell Sorter ...
Aug 9, 2025 · The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell ...Missing: hematology pre-
[35]
Microfluidic Cell Sorting: A Review of the Advances in the ... - NIH
In this review, we examine the breadth of microfluidic cell sorting technologies, while focusing on those that offer the greatest potential for translation ...
[36]
Advances in high-throughput single-cell microtechnologies
This review will focus specifically on high throughput technologies recently developed for the purpose of analysis and isolation of single cells from ...
[37]
Practical Considerations for Single-Cell Genomics - PMC - NIH
In this overview article, we describe a variety of popular single-cell genomics technologies and address some common questions pertaining to study design.
[38]
Cell Sorting: Automated Separation of Mammalian Cells ... - Science
A system for high-speed sorting of fluorescent cells was able to sort mouse spleen cells from Chinese hamster ovarian cells after development of ...Missing: original | Show results with:original<|separator|>
[39]
How a Flow Cytometer Works | Thermo Fisher Scientific - US
This specialized flow cytometer is called a fluorescence activated cell sorter (FACS) ... There are 3 main components of a flow cytometer—the fluidics, optics ...Introduction To Flow... · Table 1. Comparing Flow... · Potential Applications For...
[40]
High-throughput, low-loss, low-cost, and label-free cell separation ...
Apr 13, 2017 · FACS can sort up to 50,000 cells per second, with higher rates achievable at the cost of purity. Finally, magnetically activated cell ...
[41]
Fluorescence Fundamentals | Thermo Fisher Scientific - US
§ EC (units: cm-1 M-1) is defined by the Beer-Lambert law A=EC•c•l, where A = absorbance, c = molar concentration, l = optical pathlength. EC values at the ...Missing: equation | Show results with:equation
[42]
Magnetic cell separation | Miltenyi Biotec | USA
The strong magnetic field within a MACS Column allows for minimal cell labeling with nano-sized beads, preventing epitope saturation and non-specific labeling. ...Missing: superparamagnetic | Show results with:superparamagnetic
[43]
High gradient magnetic cell separation with MACS - Miltenyi - 1990
A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed ...
[44]
Basic principle of magnetic cell separation | Miltenyi Biotec | USA
Separate labeled cells from unlabeled ones using a MACS Column. The amplified magnetic field efficiently captures labeled cells while enabling the smooth flow ...Missing: immunomagnetic history
[45]
Fundamentals and Application of Magnetic Particles in Cell Isolation ...
Magnetic sorting using magnetic beads has become a routine methodology for the separation of key cell populations from biological suspensions.
[46]
MACS® MicroBeads - magnetic beads for gentle cell isolation
Our unique combination of nano-sized MACS MicroBeads and specialized MACS Columns enables gentle cell isolation with minimal labeling.
[47]
Small but mighty: How the MACS®‐technology based on nanosized ...
Jun 25, 2010 · Magnetic cell sorting has evolved from the original fluorescence activated cell sorting (FACS)-support technology to a real alternative, for ...
[48]
Basic Principles and Recent Advances in Magnetic Cell Separation
Miltenyi Biotech has recently commercialized a technology based on releasable antibodies enabling the removal of both magnetic beads and antibody fragments ...
[49]
[PDF] Sorting out the benefits - Miltenyi Biotec
• High cell throughput: 10⁹–10¹⁰ cells/hour. • Rapid isolation of rare cells: reduced sorting time for maintenance of full functionality. • Unique ...
[50]
Marker-specific sorting of rare cells using dielectrophoresis - PNAS
To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in ...
[51]
Acoustophoretic Sorting of Viable Mammalian Cells in a Microfluidic ...
We report the first use of ultrasonic acoustophoresis for the label-free separation of viable and nonviable mammalian cells within a microfluidic device.
[52]
Continuous Particle Separation Through Deterministic Lateral ...
We report on a microfluidic particle-separation device that makes use of the asymmetric bifurcation of laminar flow around obstacles.
[53]
Pinched Flow Fractionation: Continuous Size Separation of Particles ...
In the present study, we propose a new and simple method for the size separation of particles, pinched flow fractionation (PFF), in a microchannel that has a ...Principle · Experimental Section · Conclusions · Supporting Information Available
[54]
Continuous inertial focusing, ordering, and separation of particles in ...
We observe that flowing particles migrate across streamlines in a continuous, predictable, and accurate manner in microchannels experiencing laminar flows.
[55]
Cell Separation and Cell Isolation Methods
Density gradient centrifugation is an inexpensive cell separation technique but has limited specificity, low purity, and low throughput. In addition, even ...
[56]
Fractionation of cells and subcellular particles with Percoll
Percoll is used in density gradient centrifugation to separate cells and subcellular particles, reducing convection and separating by size or density.
[57]
Methods of Granulocyte Isolation from Human Blood and Labeling ...
The best method of granulocyte isolation was Percoll with Ficoll, which showed 95.92% purity and 94% viability. After labeling with M-SPIONs, the granulocytes ...Missing: throughput | Show results with:throughput
[58]
Zonal Centrifugation - an overview | ScienceDirect Topics
Rate-zonal centrifugation is used to separate two or more populations of particles from each other by virtue of a difference in their sedimentation rates. The ...
[59]
Separation of cells by velocity sedimentation - Semantic Scholar
Centrifugation in density gradients, exploiting differences in sedimentation rate and in buoyant density, has achieved the separation of myeloid cells from ...<|separator|>
[60]
Principles of Continuous Flow Centrifugation - Beckman Coulter
The sedimentation coefficient of the particles to be collected governs the selection of flow rate and operating speed. Small particles require either a higher ...To Process Material In... · Rotors, Cores, And Their... · Bibliography Of Applications
[61]
An Optimized and Versatile Counter-Flow Centrifugal Elutriation ...
Apr 20, 2021 · Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior.
[62]
What is Centrifugal Elutriation - Beckman Coulter
Centrifugal elutriation is a gentle process that is faster than flow cytometry, quickly and efficiently processing large numbers of purified cells.Missing: sorting review
[63]
Optimization of single-cell plate sorting for high throughput ... - NIH
Dec 24, 2018 · Introduction. Index sorting is a powerful tool that links protein expression with single cell gene expression and sequencing analysis.
[64]
[PDF] BD FACSAria™ Platform Performing Index Sorting
This job aid contains instructions for performing index sorting using BD FACSDiva™ software. Review the entire job aid prior to index sorting. Review the Tips ...Missing: deposition | Show results with:deposition
[65]
Combined Single-Cell Functional and Gene Expression Analysis ...
May 21, 2015 · The integration of the genome-wide scRNA-seq analysis with the index sorting data also suggests that while functional HSCs are typically dormant ...
[66]
Single‐Cell Analysis Using Droplet Microfluidics - Wiley Online Library
Nov 26, 2019 · Droplet microfluidics has revolutionized single-cell analysis. This review provides a comprehensive overview of key breakthroughs in ...
[67]
Single-cell analysis and sorting using droplet-based microfluidics
Abstract. We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells ...Missing: review | Show results with:review
[68]
Photoactivated Selective Release of Droplets from Microwell Arrays
Jan 8, 2020 · In this report, we present photoactivated selective release of droplets from a static microwell array enabled by a photoresponsive polymer layer integrated ...
[69]
An automated system for high-throughput single cell-based breeding
Feb 1, 2013 · Each cell was transferred and released into the culture medium of an assigned well in 96- or 384-well plates (step f). ... 384-well plates at ~1 ...
[70]
Traceable impedance-based single-cell pipetting, from a research ...
Dec 17, 2021 · DispenCell is an innovative impedance-based method for single-cell isolation. DispenCell automation increases throughput, robustness and user-friendliness.
[71]
Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells ...
Aug 21, 2023 · Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when ...
[72]
New killer application for cell sorter chip | imec
Oct 2, 2019 · In numbers: it can sort 5,000 cells/sec per single microfluidic channel, with a > 90% cell sorting yield, up to 99% purity and well-preserved ...
[73]
Review The Technology and Biology of Single-Cell RNA Sequencing
May 21, 2015 · By integrating scRNA-seq with single-cell proteomics information from either FACS or CyToF (Amir el et al., 2013), it will become clear ...
[74]
Improving Single-Cell Encapsulation Efficiency and Reliability ... - NIH
Jan 15, 2020 · We found that using a neutrally buoyant suspension greatly increased the efficiency of single-cell encapsulation in microdroplets and eliminated unnecessary ...3. Materials And Methods · 3.2. Cell Culture And... · 4.3. Cell Viability And...
[75]
Enhancing single-cell encapsulation in droplet microfluidics with fine ...
Jan 2, 2024 · Low-density samples and larger channels enhanced the focusing efficiency, reduced the flow shear stress and lowered the possibility of clogging.Results · Bead Focusing And Enrichment · Cell Focusing And Enrichment
[76]
Magnetic cell sorting and flow cytometry sorting methods for ... - NIH
Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells · Abstract · INTRODUCTION.
[77]
Human CD4+CD25+CD226- Tregs Demonstrate Increased Purity ...
Current Treg isolation methods involve Fluorescence-Activated Cell Sorting (FACS) of CD4+CD25hi T cells with low to no expression of the IL-7 receptor, CD127 ( ...Treg Expansion · Treg Suppression Assays · Cd25 Expression Is Elevated...
[78]
Concise Review: Evidence for CD34 as a Common Marker for ... - NIH
In clinical practice, CD34 expression is evaluated to ensure rapid engraftment in BM transplants and can also be used as a selective marker in cell sorting to ...
[79]
Circulating tumor cells expressing cancer stem cell marker CD44 as ...
Epithelial cell adhesion molecule (EpCAM) is a marker for circulating tumor cells (CTCs) in various types of cancer, while cluster of differentiation 44 (CD44) ...
[80]
Protocol for performing pooled CRISPR-Cas9 loss-of-function screens
Mar 30, 2023 · We have performed death-based and fluorescence-activated cell sorting (FACS)-based CRISPR KO screens to identify key regulators of ferroptosis1 ...
[81]
Deep immune profiling of COVID-19 patients reveals distinct ...
High-dimensional flow cytometry and longitudinal analysis highlighted stability and fluctuations in the response.
[82]
CAR-T cell manufacturing: Major process parameters and next ...
Jan 16, 2024 · In this review, we discuss the major process parameters of autologous CAR-T cell manufacturing, as well as regulatory considerations and ongoing developments.
[83]
Human CD34+ hematopoietic stem cell hierarchy: how far are ... - NIH
... applications of transplantation, gene therapy, stem cell expansion, and tumor-cell purging. In this article, we will critically review the progress in the ...
[84]
EpCAM based capture detects and recovers circulating tumor cells ...
EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low - PMC.
[85]
Generation of human cerebral organoids with a structured outer ...
(A) FACS sorting strategy to isolate SOX2+ HOPX+ progenitor cells from LIF-treated brain organoids and test their neurogenic differentiation potential in a ...
[86]
Integration of spatial and single-cell transcriptomic data elucidates ...
Sep 6, 2021 · We apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in ...
[87]
Image-based cell sorting using focused travelling surface acoustic ...
Post-sorting analysis of the target sample showed an increase in purity from 52.6% (initial purity) to 86.3% (target purity) which corresponds to a 1.64 ...
[88]
How to Avoid Cell Stress While Cell Sorting - Bio-Rad Antibodies
Sep 24, 2020 · Inappropriate nozzle sizes can increase cell stress and decrease viability by causing inappropriate deposition and intense shear force. 4) Use ...
[89]
High-Pressure vs. Microfluidic: Gentle Cell Sorting | NanoCellect
Jan 30, 2023 · This dramatic change in pressure causes decompression shock and shear stress, leading to damaged cells with poor cell health and viability.
[90]
How Much Does A Flow Cytometer Cost in 2024? - Excedr
Jan 10, 2024 · High-End Flow Cytometers (6+ lasers, cell sorting): $500,000 to $1,000,000. These models are designed for specialized labs that need advanced ...
[91]
https://www.excedr.com/blog/how-much-does-a-flow-cytometer-cost
[92]
Characterization of aerosols produced by cell sorters and evaluation ...
The present study uses an Aerodynamic Particle Sizer, to evaluate aerosol production and containment on the FACS Aria II model of cell sorter.
[93]
Cell Sorting 2025 Trends and Forecasts 2033: Analyzing Growth ...
In stock Rating 4.8 (1,980) Jun 15, 2025 · Point-of-care cell sorting: Development of portable and user-friendly devices for cell sorting in resource-limited settings. Increased use ...
[94]
End Cross-Contamination in Flow Cytometry through Microfluidics
May 18, 2021 · Aerosols—a source of environmental contamination—are also avoided due to the nature of the flow shift method. Made from inexpensive ...
[95]
Accelerating CRISPR Workflows with Gentle Microfluidic Sorting
Nov 3, 2025 · In this study, researchers performed CRISPR editing and used single-cell sorting into 96-well plates with the WOLF Cell Sorter (with N1 ...
[96]
A facile methodology using quantum dot multiplex labels for tracking ...
Jun 27, 2019 · Herein we demonstrate that this can be achieved using a facile methodology in which quantum dots (QDs) are used to label two different plasmid ...
[97]
Imaging flow cytometry with a real-time throughput beyond ... - Nature
Feb 10, 2025 · Cells flowing at speeds up to 15 m/s are clearly imaged with a spatial resolution of 780 nm, and images of each individual cell are captured, ...