Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry

Current Protocols in Cytometry, Journal Year: 2020, Volume and Issue: 92(1)

Published: March 1, 2020

Abstract Technological advances in fluorescence flow cytometry and an ever‐expanding understanding of the complexity immune system have led to development large (20+ parameters) panels. However, as panel size increase, so does difficulty involved designing a high‐quality panel, accessing instrumentation capable accommodating numbers parameters, analyzing such high‐dimensional data. A recent advancement is spectral cytometry, which contrast conventional distinguishes full emission spectrum each fluorophore across all lasers, rather than identifying only peak emission. Fluorophores with similar maximum but distinct off‐peak signatures can therefore be accommodated within same allowing greater flexibility terms design detection. Here, we highlight specific characteristics aim guide users through process building, designing, optimizing panels using comprehensive step‐by‐step protocol. Special considerations are also given for highly overlapping dyes, logical selection optimal marker‐fluorophore assignment provided. © 2020 by John Wiley & Sons, Inc.

Language: Английский

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Plasmonic Optical Tweezers for Particle Manipulation: Principles, Methods, and Applications

ACS Nano, Journal Year: 2021, Volume and Issue: 15(4), P. 6105 - 6128

Published: April 9, 2021

Inspired by the idea of combining conventional optical tweezers with plasmonic nanostructures, a technique named (POT) has been widely explored from fundamental principles to applications. With ability break diffraction barrier and enhance localized electromagnetic field, POT techniques are especially effective for high spatial-resolution manipulation nanoscale or even subnanoscale objects, small bioparticles atoms. In addition, can be easily integrated other such as lab-on-chip devices, which results in very promising alternative high-throughput single-bioparticle sensing imaging. Despite its label-free, high-precision, high-spatial-resolution nature, it also suffers some limitations. One main obstacles is that nanostructures located over surfaces substrate, makes turn three-dimensional problem nearly two-dimensional problem. Meanwhile, operation zone limited predefined area. Therefore, target objects must delivered near structures. This review summarizes state-of-the-art delivery methods POT-based particle manipulating technique, along applications analysis/imaging, bioparticle purifying, single-atom manipulation. Future developmental perspectives discussed.

Language: Английский

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Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria

Scientific Reports, Journal Year: 2019, Volume and Issue: 9(1)

Published: Dec. 9, 2019

Abstract Despite the development of several cultivation methods, rate discovery microorganisms that are yet-to-be cultivated outpaces isolating and cultivating novel species in laboratory. Furthermore, no current technique is capable selectively specific bacterial taxa or phylogenetic groups independently morphological physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed can survive modified version standard fluorescence situ hybridization (FISH) procedure, which fixation omitted other factors, such as centrifugation buffers, optimized. also demonstrated labelled DNA probes be introduced into cells by means chemical transformation occurs. This method, call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) sort taxonomic from mock natural communities subsequently culture them. Live-FISH represents first attempt systematically optimize conditions known affect viability during FISH surviving procedure. No sophisticated probe design required, making live-FISH straightforward potentially used combination single-cell techniques for isolation microorganisms.

Language: Английский

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Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

Nature Communications, Journal Year: 2020, Volume and Issue: 11(1)

Published: Dec. 8, 2020

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which difficult to mimic in bottom-up synthetic research and limits the functionality existing protocellular platforms. There thus a clear need for general, biocompatible, accessible tool more accurately emulate this environment. Herein, we describe development discrete, membrane-bound coacervate-based platform that utilizes well-known binding motif between Ni

Language: Английский

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Murine macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing

European Journal of Immunology, Journal Year: 2018, Volume and Issue: 48(9), P. 1445 - 1455

Published: June 7, 2018

Macrophages play a critical role in the establishment of regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct initiation resolution inflammation that is essential for repair. In pathologic states where chronic prevents healing, macrophages fail transition reparative phenotype. Using murine model cutaneous we found CCR2-deficient mice (CCR2-/- ) demonstrate significantly impaired healing at all time points postinjury. Flow cytometry analysis wounds CCR2-/- WT revealed significant decrease inflammatory, Ly6CHi monocyte/macrophages wounds. We further show macrophage cytokine production decreased Adoptive transfer mT/mG into CCR2+/+ demonstrated labeled cells on days 2 4 traveled both mice. Further, adoptive restored normal likely through Taken together, these data suggest CCR2 plays recruitment repair may be therapeutically manipulated modulation CCR2.

Language: Английский

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