Pulse Width for Particle Sizing

Hoffman, R. A.

doi:10.1002/0471142956.cy0123s50

Cited by 32 publications

(31 citation statements)

References 4 publications

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“…The result of these electronic features was high sensitivity with low thresholding capabilities which were critical for dim signals and studying submicron particles. The pulse width parameter has not previously been used in many research studies but has been shown to be useful in cell cycle studies for removing G1 doublets from G2/M cells, and in some cases to detect high fluorescence objects located in a cell . In the current studies, pulse width was measured directly and not used as a calculated ratio between other signal parameters (i.e.…”

Section: Methodsmentioning

confidence: 99%

Characterization, detection, and counting of metal nanoparticles using flow cytometry

2015

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There is a need to accurately detect, characterize, and quantify nanoparticles in suspensions. This study helps to understand the complex interactions between similar types of nanoparticles. Before initiating a study of metal nanoparticles, five submicron PS beads with sizes between 200 nm and 1 mm were used to derive a reference scale that was useful in evaluating the flow cytometer for functionality, sensitivity, resolution, and reproducibility. Side scatter intensity (SSC) from metal nanoparticles was obtained simultaneously from 405 nm and 488 nm lasers. The 405 nm laser generally yielded histogram distributions with smaller CVs, less side scatter intensity, better separation indices between beads and decreased scatter differences between different sized particles compared with the 488 nm laser. Submicron particles must be diluted to 10 6 and 10 7 particles/mL before flow cytometer analysis to avoid coincidence counting artifacts. When particles were too concentrated the following occurred: swarm, electronic overload, coincidence counting, activation of doublet discrimination and rejection circuitry, increase of mean SSC histogram distributions, alterations of SSC and pulse width histogram shape, decrease and fluctuations in counting rate and decrease or elimination of particulate water noise and 1 mm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1mm counting beads. Sequential dilutions of metal nanoparticles in a 1 mm counting bead suspension helped determine the diluted concentration needed for flow cytometer analysis. It was found that the original concentrated nanoparticle samples had to be diluted, between 1:10,000 and 1:100,000, before characterization by flow cytometry. The concentration of silver or gold nanoparticles in the undiluted sample were determined by comparing them with a known concentration (1.9 3 10 6 beads/mL) of 1 mm polystyrene reference beads. Published 2015 Wiley Periodicals Inc., on behalf of ISAC

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Section: Methodsmentioning

confidence: 99%

Characterization, detection, and counting of metal nanoparticles using flow cytometry

2015

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“…The size standards need to have a refractive index similar to the sample material, which poses a challenge for protein particles as most size standards such as polystyrene beads show refractive indices much higher than protein particles. Size determination of labeled material is also possible based on the pulse width of the fluorescence signal after calibration with fluorescent size standards 110. Compared with other light scattering techniques such as DLS or SLS, FAPS analyzes particles individually and therefore shows size distributions of higher resolution 67.…”

Section: Light Scattering Methodsmentioning

confidence: 99%

Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods

Zölls¹,

Tantipolphan²,

Wiggenhorn³

et al. 2012

Journal of Pharmaceutical Sciences

201

159

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“…The use of the height and width parameters of fluorochromes was first demonstrated to distinguish cells and particles of different sizes (Hoffman, 2009). This was subsequently expanded to examine mammalian cells, where PulSA was used to detect aggregates of the Huntington's protein in cells, a hallmark of the disease (Ramdzan et al, 2012).…”

Section: Of 21mentioning

confidence: 99%

Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues

2020

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One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web‐like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large‐scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET‐associated markers can be nonspecific, time‐consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse‐shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA‐diffuse neutrophils is found in cell populations after exposure to NET‐inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large‐scale experiments. © 2020 Wiley Periodicals LLC. Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow Alternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytes Alternate Protocol 2: Detection of nuclear decondensation in neutrophils from blood Support Protocol 1: Cryopreservation and defrosting of samples Support Protocol 2: Paraformaldehyde fixation of samples

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Pulse Width for Particle Sizing

Cited by 32 publications

References 4 publications

Characterization, detection, and counting of metal nanoparticles using flow cytometry

Characterization, detection, and counting of metal nanoparticles using flow cytometry

Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods

Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues

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