scholarly journals Culture and Characterization of Microglia from the Adult Murine Retina

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Gayathri Devarajan ◽  
Mei Chen ◽  
Elizabeth Muckersie ◽  
Heping Xu
Keyword(s):  
Single Cell ◽  
Proinflammatory Cytokines ◽  
Cultured Cells ◽  
Calf Serum ◽  
Culture Conditions ◽  
Simple Method ◽  
Mhc Ii ◽  
Adult Mice ◽  
Retinal Microglia ◽  
And Function

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells.Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay.Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of0.75×106cells per cm2in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-αand express CD86, CD40, and MHC-II.Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.

scholarly journals Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production.

1981 ◽  
Vol 90 (2) ◽  
pp. 332-338 ◽  
Author(s):  
R P Mecham ◽  
G Lange ◽  
J Madaras ◽  
B Starcher
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Elastic Fiber ◽  
Calf Serum ◽  
Phenotypic Expression ◽  
Fetal Tissue ◽  
Culture Conditions ◽  
Population Doubling

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.

scholarly journals MORPHOLOGY AND FUNCTION OF CELLS OF HUMAN EMBRYONIC LIVER IN MONOLAYER CULTURE

1971 ◽  
Vol 50 (1) ◽  
pp. 222-231 ◽  
Author(s):  
D. Montgomery Bissell ◽  
Jeremiah G. Tilles
Keyword(s):  
Cultured Cells ◽  
Fetal Liver ◽  
Calf Serum ◽  
Liver Cells ◽  
Cultured Liver Cells ◽  
Subsequent Loss ◽  
Fetal Liver Cells ◽  
And Function ◽  
And Storage ◽  
Human Fetal Liver Cells

A system for culturing human fetal liver cells in monolayers is described and the effects of various conditions of growth on the morphology and function of the cultured cells are presented. The addition of 10% calf serum or 1% human serum to the growth medium accelerated the proliferation of the liver cells, with subsequent loss of characteristic morphology and specific functional activity. In the absence of serum, the cultured liver cells retained their morphology and their function for at least 4 wk, as evidenced by secretion of serum albumin and storage of glycogen and iron.

A potassium permanganate fixative for membranes in sections

1990 ◽  
Vol 48 (3) ◽  
pp. 722-723
Author(s):  
Jindan Song
Keyword(s):  
Potassium Permanganate ◽  
Cultured Cells ◽  
Calf Serum ◽  
Trisodium Citrate ◽  
Membrane System ◽  
Adenocarcinoma Cell Line ◽  
Mouse Embryo Fibroblast ◽  
African Green Monkey Kidney ◽  
Adenocarcinoma Cell ◽  
Green Monkey

Potassium permanganate has been used as a fixative for the botanical specimen and membrane system in thin section by Glauert (1975). A new potassium permanganate fixative ( Trisodium citrate 60mM, Potassium chloride 25mM, Magnesium chloride 35mM, and Potassium permanganate 125mM ) for localizing membranous system in whole_mount cultured cells with standard trasmission electron microscopy and phase_contrast microscopy has been developed). Here, we report that using this new potassium permanganate fixative for membranous system in sections.Cultured cells, CV_1 (African green monkey kidney epithelial cells), Balb/c 3T3 ( Mouse embryo fibroblast ) and MCF_7 (Human adenocarcinoma cell line) were used for this study. All cells were grown on 35mm plastic dishes in DME medium containing 5% calf serum at 37 c with 100% humidity and 5% CO2. Using the potassium permanganate fixative to fix the cells for about 7 minutes. After fixation, the cells were dehydrated in a graded series of ethanol.

A Mucin-Specific Protease Enables Molecular and Functional Analysis of Human Cancer-Associated Mucins

2018 ◽  
Author(s):  
Stacy A. Malaker ◽  
Kayvon Pedram ◽  
Michael J. Ferracane ◽  
Elliot C. Woods ◽  
Jessica Kramer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Human Cancer ◽  
Glycosylation Sites ◽  
Bacterial Protease ◽  
Specific Protease ◽  
To Come ◽  
And Function

<div> <div> <div> <p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular surfaces and are important for human health, especially in the context of carcinomas. However, the molecular mechanisms by which aberrant mucin structures lead to tumor progression and immune evasion have been slow to come to light, in part because methods for selective mucin degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis, and computational peptide docking to demonstrate that a bacterial protease, called StcE, cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure- based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9 receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding receptors. </p> </div> </div> </div>

scholarly journals The Drosophila engrailed and invected Genes: Partners in Regulation, Expression and Function

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 893-906 ◽  
Author(s):  
Elizabeth Gustavson ◽  
Andrew S Goldsborough ◽  
Zehra Ali ◽  
Thomas B Kornberg
Keyword(s):  
Expression Pattern ◽  
Cultured Cells ◽  
Regulatory Region ◽  
Embryonic Lethality ◽  
Wild Type ◽  
Terminal Portion ◽  
Coding Sequence ◽  
Differential Effects ◽  
And Function ◽  
Redundant Functions

Abstract We isolated and characterized numerous engrailed and invected alleles. Among the deficiencies we isolated, a mutant lacking invected sequences was viable and phenotypically normal, a mutant lacking engrailed was an embryo lethal and had slight segmentation defects, and a mutant lacking both engrailed and invected was most severely affected. In seven engrailed alleles, mutations caused translation to terminate prematurely in the central or C-terminal portion of the coding sequence, resulting in embryonic lethality and segmentation defects. Both engrailed and invected expression declined prematurely in these mutant embryos. In wild-type embryos, engrailed and invected are juxtaposed and are expressed in essentially identical patterns. A breakpoint mutant that separates the mgrailed and invected transcription units parceled different aspects of the expression pattern to engrailed or invected. We also found that both genes cause similar defects when expressed ectopically and that the protein products of both genes act to repress transcription in cultured cells. We propose that the varied phenotypes of the engrailed alleles can be explained by the differential effects these mutants have on the combination of engrailed and invected activities, that engrailed and invected share a regulatory region, and that they encode redundant functions.

Evaluation of a Domestic Steam Disinfector-Dryer Device for Disinfection of Health Care Workers’ Identification Lanyards

2021 ◽  
pp. 216507992110126
Author(s):  
Beverley C. Millar ◽  
John E. Moore
Keyword(s):  
Health Care ◽  
Health Care Workers ◽  
Culture Conditions ◽  
Broth Culture ◽  
Culture Methods ◽  
Simple Method ◽  
Diverse Range ◽  
Nosocomial Pathogens ◽  
Care Workers ◽  
Test Organisms

Background Fabric lanyards are commonly worn by health care workers (HCWs) and are known to harbor infectious organisms and contribute to the transmission of infection to HCWs and patients. A diverse range of nosocomial pathogens have been found on lanyards, but there are very few studies describing how to successfully disinfect lanyards to break the chain of transmission. Recently, a steam disinfector-dryer device has come on the market, which performs rapid disinfection against nosocomial pathogens and also dries the contents of the device. It was the aim of this study to evaluate steam disinfection-drying as a method to eliminate pathogens from lanyards. Methods Thirty-eight strips of new, unused, and autoclaved polyester neck lanyards (4 × 2 cm) were inoculated with 30 (12 Gram-positive + 18 Gram-negative) bacteria and one yeast organism. The inoculated lanyard fabric (five organisms per lanyard strip) was placed into a steam disinfector-dryer device and disinfected for 5 minutes and dried for 30 minutes, in accordance with the manufacturer’s instructions. Following disinfection and drying, the presence of viable organisms on lanyard fabric was evaluated using enhanced microbiological broth culture methods for 48 hours. Control lanyard strips were treated with organisms and left at room temperature without undergoing disinfection and drying procedures. Findings Steam disinfection-drying eradicated all test organisms from treated lanyards, with no culturable organisms detected following disinfection-drying, even when employing enhanced bacteriological culture conditions. All test organisms remained viable on the control lanyards. Conclusion/Application to Practice Steam disinfection-drying offers a simple method of decontaminating lanyards, producing dry lanyards for immediate reuse. Occupational health practitioners and hospitals should consider assessing the feasibility of adopting this method in their settings to aid in breaking the chain of transmission of nosocomial pathogens via contaminated lanyards.

scholarly journals The long noncoding RNA Synage regulates synapse stability and neuronal function in the cerebellum

2021 ◽  
Author(s):  
Fei Wang ◽  
Qianqian Wang ◽  
Baowei Liu ◽  
Lisheng Mei ◽  
Sisi Ma ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Cerebellar Atrophy ◽  
Synaptic Activity ◽  
Motor Dysfunction ◽  
Cerebellar Development ◽  
Full Spectrum ◽  
Density Reduction ◽  
Adult Mice ◽  
Synapse Density ◽  
And Function

AbstractThe brain is known to express many long noncoding RNAs (lncRNAs); however, whether and how these lncRNAs function in modulating synaptic stability remains unclear. Here, we report a cerebellum highly expressed lncRNA, Synage, regulating synaptic stability via at least two mechanisms. One is through the function of Synage as a sponge for the microRNA miR-325-3p, to regulate expression of the known cerebellar synapse organizer Cbln1. The other function is to serve as a scaffold for organizing the assembly of the LRP1-HSP90AA1-PSD-95 complex in PF-PC synapses. Although somewhat divergent in its mature mRNA sequence, the locus encoding Synage is positioned adjacent to the Cbln1 loci in mouse, rhesus macaque, and human, and Synage is highly expressed in the cerebella of all three species. Synage deletion causes a full-spectrum cerebellar ablation phenotype that proceeds from cerebellar atrophy, through neuron loss, on to synapse density reduction, synaptic vesicle loss, and finally to a reduction in synaptic activity during cerebellar development; these deficits are accompanied by motor dysfunction in adult mice, which can be rescued by AAV-mediated Synage overexpression from birth. Thus, our study demonstrates roles for the lncRNA Synage in regulating synaptic stability and function during cerebellar development.

scholarly journals CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos

2016 ◽  
Vol 27 (6) ◽  
pp. 464-475 ◽  
Author(s):  
Evguenia Kouranova ◽  
Kevin Forbes ◽  
Guojun Zhao ◽  
Joe Warren ◽  
Angela Bartels ◽  
...  
Keyword(s):  
Single Cell ◽  
Cultured Cells ◽  
Targeting Delivery

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

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