scholarly journals A method for incorporating macromolecules into adherent cells.

1984 ◽  
Vol 98 (4) ◽  
pp. 1556-1564 ◽  
Author(s):  
P L McNeil ◽  
R F Murphy ◽  
F Lanni ◽  
D L Taylor
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Cell Types ◽  
Stress Fibers ◽  
Human Neutrophils ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Simple Method ◽  
Fluorescent Molecules ◽  
Scrape Loading

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.

A simple method for incorporating aequorin into mammalian cells

1986 ◽  
Vol 251 (2) ◽  
pp. C323-C326 ◽  
Author(s):  
A. B. Borle ◽  
C. C. Freudenrich ◽  
K. W. Snowdowne
Keyword(s):  
Cardiac Myocytes ◽  
Mammalian Cells ◽  
Kidney Cells ◽  
Simple Method ◽  
Scrape Loading

A simple method for incorporating aequorin into mammalian cells to measure cytosolic ionized Ca2+ is described and compared with scrape loading and hypoosmotic treatment (HOST). The procedure consists of incubating the cells for 10 min and centrifuging them at 200 g for 30 s in the presence of aequorin. This method incorporates the same amount of photoprotein as scrape loading but 70% less than HOST. Cytosolic ionized Ca2+ has been measured in hepatocytes, kidney cells and tubules, macrophages, and cardiac myocytes loaded with aequorin by this new procedure.

scholarly journals A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface

2020 ◽  
Vol 68 (7) ◽  
pp. 473-489
Author(s):  
Anna Bäckström ◽  
Laura Kugel ◽  
Christian Gnann ◽  
Hao Xu ◽  
Joseph E. Aslan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Spatial Information ◽  
Protein Localization ◽  
Cell Types ◽  
Human Platelets ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Suspension Cells

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

scholarly journals Cell Adhesion Promotes Ebola Virus Envelope Glycoprotein-Mediated Binding and Infection

2008 ◽  
Vol 82 (14) ◽  
pp. 7238-7242 ◽  
Author(s):  
Derek Dube ◽  
Kathryn L. Schornberg ◽  
Tzanko S. Stantchev ◽  
Matthew I. Bonaparte ◽  
Sue E. Delos ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Ebola Virus ◽  
Cell Spreading ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Receptor Binding Domain ◽  
Virus Envelope ◽  
Nonadherent Cells

ABSTRACT Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

scholarly journals Silver Nanoparticles Induce Neutrophil Extracellular Traps Via Activation of PAD and Neutrophil Elastase

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 317
Author(s):  
HanGoo Kang ◽  
Jinwon Seo ◽  
Eun-Jeong Yang ◽  
In-Hong Choi
Keyword(s):  
Silver Nanoparticles ◽  
Neutrophil Elastase ◽  
Mammalian Cells ◽  
Antimicrobial Properties ◽  
Neutrophil Extracellular Traps ◽  
Arginine Deiminase ◽  
Human Neutrophils ◽  
Extracellular Traps ◽  
Peptidyl Arginine Deiminase ◽  
Key Factor

Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Production and Analysis of High Resolution Polymer Replicas of Fibrillar Collagen

1999 ◽  
Vol 5 (S2) ◽  
pp. 398-399
Author(s):  
P. Sims ◽  
B. Todd ◽  
S. Eppell ◽  
T. Li ◽  
K. Park ◽  
...  
Keyword(s):  
Cellular Response ◽  
Physical Nature ◽  
Cell Types ◽  
Artificial Substrates ◽  
Adherent Cell ◽  
Fibrillar Collagen ◽  
New Materials ◽  
Substrate Interactions ◽  
Number Of Factors ◽  
Cell Substrate

Adherent cells generally construct the immediate substrate upon which they reside. This may occur via synthesis and secretion of new materials and/or by rearrangement and modification of existing substrate. The response of adherent cell types to an existing substrate can be influenced by a number of factors which include both the chemical and physical nature of the substrate. Cell adhesion, proliferation, differentiation and death can all be substrate dependent. Much effort has been directed toward chemical modification of substrates to regulate one or more of the parameters noted above. A significant, but somewhat smaller, degree of attention has been paid to the effects of the topography and microtopography on the cell response to substrate materials. Studies to date strongly suggest the topography is a significant factor in cell-substrate interactions. As noted above, it is most probable that both the chemistry and the structure of a substrate simultaneously influence the cellular response. However we wished to determine, particularly for artificial substrates, the role which microtopography can play in cell-substrate interactions.

Inactivation of Surface-adherent Listeria monocytogenes Hypochlorite and Heat

1991 ◽  
Vol 54 (1) ◽  
pp. 4-6 ◽  
Author(s):  
SHIN-HO LEE ◽  
JOSEPH F. FRANK
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Cell Population ◽  
Incubation Time ◽  
Cell Populations ◽  
Adherent Cell ◽  
Adherent Cells ◽  
D Cells

Inactivation by hypochlorite of Listeria monocytogenes cells adherent to stainless steel was determined. Adherent cell populations were prepared by incubating stainless steel slides with a 24 h culture of L. monocytogenes for 4 h at 21°C. Adherent microcolonies were prepared by growing L. monocytogenes on stainless steel slides submerged in a 1:15 dilution of tryptic soy broth at 21°C. The slides were then rinsed and transferred to fresh sterile broth every 2 d with a total incubation time of 8 d. Although the 4 h and 8 d adherent populations were at similar levels, 8 d adherent cells were over 100 times more resistant than the 4 h adherent cell population when exposed to 200 ppm hypochlorite for 30 s. When stainless steel slides containing adherent cells were heated at 72°C both adherent cell populations were inactivated after 1 min. Detectable numbers of L. monocytogenes remained on stainless steel slides after treatment at 65°C for 3 min when adherent 8 d cells were tested but not when adherent 4 h cells were used.

Effects of red grape juice polyphenols in NADPH oxidase subunit expression in human neutrophils and mononuclear blood cells

2009 ◽  
Vol 102 (8) ◽  
pp. 1125-1135 ◽  
Author(s):  
Alberto Dávalos ◽  
Gema de la Peña ◽  
Carolina C. Sánchez-Martín ◽  
M. Teresa Guerra ◽  
Begoña Bartolomé ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Red Wine ◽  
Grape Juice ◽  
Cell Types ◽  
Human Neutrophils ◽  
Superoxide Anion Production ◽  
Oxidase Subunit ◽  
Subunit Expression ◽  
Red Grape Juice ◽  
Red Grape

The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2′7′-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

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