Single-cell lysis for visual analysis by electron microscopy

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.

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On-Chip Single-Cell Lysis for Extracting Intracellular Material

Japanese Journal of Applied Physics ◽

10.1143/jjap.46.6410 ◽

2007 ◽

Vol 46 (9B) ◽

pp. 6410-6414 ◽

Cited By ~ 15

Author(s):

Norifumi Ikeda ◽

Nobuaki Tanaka ◽

Yasuko Yanagida ◽

Takeshi Hatsuzawa

Keyword(s):

Single Cell ◽

Cell Lysis ◽

On Chip

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Picoliter liquid handling at gas/liquid interface by surface and geometry control in a micro-nanofluidic device

Journal of Micromechanics and Microengineering ◽

10.1088/1361-6439/ac4006 ◽

2021 ◽

Author(s):

Kyojiro Morikawa ◽

Shin-ichi Murata ◽

Y Kazoe ◽

Kazuma Mawatari ◽

Takehiko Kitamori

Keyword(s):

Surface Tension ◽

Single Cell ◽

Single Cell Analysis ◽

Cell Lysis ◽

Fused Silica ◽

Liquid Interface ◽

Nanofluidic Devices ◽

Fluidic Control ◽

Lysis Buffer ◽

Nanofluidic Device

Abstract In micro- and nanofluidic devices, highly precise fluidic control is essential. Conventional mechanical valves in microchannels and nanochannels have size limitations, whereas hydrophobic (Laplace) valves are generally difficult to use for low-surface-tension liquids. In the present study, we developed a method for handling picoliter volumes of low-surface-tension liquids in a micro-nanofluidic device. The proposed Laplace valve is based on the pinning effect. A fused silica micro-nanofluidic device that includes a picoliter chamber whose geometry was designed to induce capillary pinning was designed and fabricated. The measured Laplace pressure of a lysis buffer (surfactant) was consistent with the calculated pressure, indicating successful fabrication and hydrophobic surface modification. The working principle of the Laplace valve was verified. The Laplace valve maintained the lysis buffer at the gas/liquid interface for 60 min, which is sufficiently long for cell lysis operations. Finally, replacement of liquids in the picoliter chamber using the valve was demonstrated. The proposed method will contribute to basic technologies for fluidic control in micro- and nanofluidic devices, and the proposed Laplace valve can be used for low-surface-tension liquids. In addition, the developed valve and picoliter chamber can be utilized for the interface in single-cell lysis, which will facilitate the development of single-cell analysis devices.

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Nanothorn Filter-Facilitated Online Cell Lysis for Rapid and Deep Intracellular Profiling by Single-Cell Mass Spectrometry

Analytical Chemistry ◽

10.1021/acs.analchem.1c03529 ◽

2021 ◽

Author(s):

Shu-Ting Xu ◽

Cheng Yang ◽

Xiu-Ping Yan

Keyword(s):

Mass Spectrometry ◽

Single Cell ◽

Cell Mass ◽

Cell Lysis ◽

Cell Mass Spectrometry

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Changes Induced by Pre-Fixation in Polycation Bacteriolysis and Surface Alterations Matching the Model of Wall Picnosis in Cell Lysis

Zeitschrift für Naturforschung C ◽

10.1515/znc-1980-9-1023 ◽

1980 ◽

Vol 35 (9-10) ◽

pp. 805-810

Author(s):

Alexandru Popescu ◽

Stefan Antohi ◽

Stefan Trasculescu ◽

Adriana Aurescu ◽

Nicolae Manolescu ◽

...

Keyword(s):

Electron Microscopy ◽

Osmium Tetroxide ◽

Cell Lysis ◽

Cell Sensitivity ◽

Lytic Effect ◽

Shape And Size ◽

Scaning Electron Microscopy

Abstract Pretreatments of B. subtilis and S. aureus cells with lower concentrations of fixative agents, led to modifications in bacteriolytic effect exerted by polyarginine and protamine: Glutaraldehyde blocked polycation bacteriolysis while formaldehyde and osmium tetroxide (OSO4) having no influence on polyarginine action, increased constantly the cell sensitivity to protamine in lower doses otherwise nonlytic; the sensitizing action also resulted in the extension of protamine bacteriolytic pattern including several staphylococcal strains; higher bacteriolytic doses of protamine were contrastively unable to lyse OSO4 prefixed cells and gave an inconstant lytic value with formaldehyde treated bacteria. With higher concentrations, OSO4 preserved intactly its sensitizing action while formaldehyde displayed a decrease in its ability to sensitize B. subtilis cells to the lytic effect of protamine. Scaning electron microscopy of polycation treated cells showed prelytic lesions as surface granulations, shape and size modifications and cell splits. The interpretation of the results in terms of intra-and intermolecular adducts accompanied by con formational changes in surface macromolecules is discussed. It is concluded that the results match the model of polycation bacteriolysis by wall multizonal picnosis leading to surface splits and thereby triggering cell-lysis.

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Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01410-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2223-2225 ◽

Cited By ~ 89

Author(s):

Nicole Cotroneo ◽

Robert Harris ◽

Nancy Perlmutter ◽

Terry Beveridge ◽

Jared A. Silverman

Keyword(s):

Electron Microscopy ◽

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Division ◽

Bactericidal Activity ◽

Membrane Integrity ◽

Cell Lysis

ABSTRACT The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

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Low-Dose Streptozocin-Induced Diabetes in Mice: Electron Microscopy Reveals Single-Cell Insulitis Before Diabetes Onset

Diabetes ◽

10.2337/diab.37.1.21 ◽

1988 ◽

Vol 37 (1) ◽

pp. 21-27 ◽

Cited By ~ 81

Author(s):

V. Kolb-Bachofen ◽

S. Epstein ◽

U. Kiesel ◽

H. Kolb

Keyword(s):

Electron Microscopy ◽

Single Cell ◽

Low Dose ◽

Diabetes Onset ◽

Diabetes In Mice

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Single-cell transcriptomics reveals a conserved metaplasia program in pancreatic injury

10.1101/2021.04.09.439243 ◽

2021 ◽

Author(s):

Zhibo Ma ◽

Nikki K Lytle ◽

Bob Chen ◽

Nidhi Jyotsana ◽

Sammy Weiser Novak ◽

...

Keyword(s):

Electron Microscopy ◽

Single Cell ◽

Disease Progression ◽

Tissue Injury ◽

Yellow Fluorescent Protein ◽

Acinar Cells ◽

Enteroendocrine Cells ◽

Intermediate Cell ◽

Chronic Injury ◽

Tuft Cell

BACKGROUND & AIMS: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from genetically wild type ADM, the associated transcriptional changes, and to identify markers of disease progression. METHODS: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Single-cell trajectories were generated. Data were compared to gastric metaplasia and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTS: scRNA-seq of ADM revealed emergence of a mucin/ductal population (Muc6+, Tff2+) resembling gastric pyloric, gland-base cells. Lineage trajectories suggest that this pyloric metaplasia is an intermediate cell identity between acinar cells and the generation of metaplastic tuft and enteroendocrine cells (EECs). 3-D electron microscopy demonstrates that all identified lineages populate ADM lesions. EECs exhibit substantial heterogeneity, including emergence of enterochromaffin (5-HT+) and delta (SST+) cells. Human pancreatitis shows a similar pyloric metaplasia phenotype and a conserved transcriptional program. CONCLUSIONS: Under conditions of chronic injury, acinar cells undergo pyloric metaplasia to mucinous progenitor-like populations, some of which can then seed disparate tuft cell and EEC lineages. EEC subtypes are diverse with the potential to direct disease progression. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases. Keywords: plasticity, pancreatitis, paligenosis, SPEM, Tuft cells, Enteroendocrine cells

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Pilot investigation into osteotome hard surface coating and cutting-edge degradation

10.21203/rs.3.rs-16381/v1 ◽

2020 ◽

Author(s):

David White ◽

Jim Bartley ◽

Christopher Whittington ◽

Lorenzo M. G. Garcia ◽

Kaushik Chand ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Titanium Nitride ◽

Visual Inspection ◽

Visual Analysis ◽

Electroless Nickel ◽

Cutting Edge ◽

Edge Sharpness ◽

Blade Edge ◽

Scanning Electron

Abstract BackgroundOsteotomes are bone cutting tools commonly reused in orthopedic surgical procedures. Despite undergoing rigorous cleaning, visual inspection and sterilization procedures between every use, the condition of the cutting blade edge is commonly not qualitatively assessed. Subjective feedback from surgeons suggests a large variation in osteotome cutting edge sharpness is found during use. This study seeks to investigate the retention of osteotome cutting-edge sharpness by comparing the wear resistance of as-supplied, electroless nickel, and titanium nitride coated osteotomes following a series of bone cutting tests.MethodsChanges in edge sharpness were assessed using visual inspection, depth penetration testing that quantified change in the blade sharpness index and scanning electron microscopy visual analysis. Visual inspection of each osteotome blade edge was then compared to qualitative blade sharpness index measurement.ResultsAfter use, no cutting-edge damage or change in blade sharpness was detected by visual examination of all three osteotomes however the as-supplied osteotome demonstrated 50% loss of blade sharpness index compared to 30% and 15% reduction for the electroless nickel and titanium nitride coated osteotomes respectively. This finding was supported by scanning electron microscopy evaluation that found greater mechanical damage had occurred along the cutting-edge of the as-supplied osteotome compared to the two coated with wear resistant materials.ConclusionsThe rapid loss of blade sharpness found in the as-supplied osteotome supports the degradation in cutting performance frequently reported by surgeons. The findings from this study demonstrates blade sharpness index better detects cutting edge wear compared to visual inspection. Results from this pilot study also suggest the coating of osteotomes in hard-wearing biocompatible materials assists in retaining cutting edge sharpness over multiple uses. Further study using a larger sample size is required to validate these findings.

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