A modified high-yield method for primary culture of rat retinal microglial cells

ABSTRACT To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-α secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1β secreted from microglial cells cocultured with A. culbertsonitrophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-α and IL-1β from microglial cells.

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Apoptosis of Primary-Culture Rat Microglial Cells Induced by Pathogenic Acanthamoeba spp

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.7.3.510-514.2000 ◽

2000 ◽

Vol 7 (3) ◽

pp. 510-514 ◽

Cited By ~ 12

Author(s):

Ho-Joon Shin ◽

Myung-Soo Cho ◽

Hyung-Il Kim ◽

Millina Lee ◽

Sun Park ◽

...

Keyword(s):

Primary Culture ◽

Dna Fragmentation ◽

Chromatin Condensation ◽

Background Level ◽

Microglial Cells ◽

Apoptotic Bodies ◽

Dna Ladder ◽

Nuclear Condensation ◽

Electron Microscopic ◽

Acanthamoeba Culbertsoni

ABSTRACT To determine whether trophozoites and lysates of pathogenicAcanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.

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Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02478-y ◽

2020 ◽

Vol 472 (12) ◽

pp. 1769-1782 ◽

Cited By ~ 1

Author(s):

Stephan Leisengang ◽

Franz Nürnberger ◽

Daniela Ott ◽

Jolanta Murgott ◽

Rüdiger Gerstberger ◽

...

Keyword(s):

Spinal Cord ◽

Transcription Factors ◽

Substance P ◽

Primary Culture ◽

Primary Cultures ◽

Somatosensory System ◽

Microglial Cells ◽

Prostaglandin E ◽

Short Term ◽

Stimulation Of

AbstractOne maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 μg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 μg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

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Anti-oxidant and anti-inflammatory activities of macelignan in murine hippocampal cell line and primary culture of rat microglial cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2005.04.036 ◽

2005 ◽

Vol 331 (4) ◽

pp. 1264-1269 ◽

Cited By ~ 76

Author(s):

Da-Qing Jin ◽

Chol Seung Lim ◽

Jae Kwan Hwang ◽

Ilho Ha ◽

Jung-Soo Han

Keyword(s):

Primary Culture ◽

Cell Line ◽

Microglial Cells ◽

Hippocampal Cell ◽

Anti Oxidant ◽

Anti Inflammatory

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Harvest and primary culture of the murine aldosterone-sensitive distal nephron

AJP Renal Physiology ◽

10.1152/ajprenal.00668.2014 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1306-F1315 ◽

Cited By ~ 4

Author(s):

Mariana Labarca ◽

Jonathan M. Nizar ◽

Elisabeth M. Walczak ◽

Wuxing Dong ◽

Alan C. Pao ◽

...

Keyword(s):

Primary Culture ◽

Magnetic Beads ◽

Collecting Duct ◽

Short Circuit ◽

Distal Convoluted Tubule ◽

High Yield ◽

Kidney Cortex ◽

Cortical Collecting Duct ◽

Distal Nephron ◽

Anion Secretion

The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm2) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.

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Quality films from carbon fiber evaporation for thorough coverage of TEM grids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100104157 ◽

1988 ◽

Vol 46 ◽

pp. 418-419

Author(s):

N. Tempel ◽

M. C. Ledbetter

Keyword(s):

Electron Microscopy ◽

High Resolution Electron Microscopy ◽

Carbon Films ◽

Low Noise ◽

Vacuum Evaporation ◽

High Yield ◽

Good Adhesion ◽

Noise Structure ◽

Resolution Electron ◽

Carbon Foils

Carbon films have been a support of choice for high resolution electron microscopy since the introduction of vacuum evaporation of carbon. The desirable qualities of carbon films and methods of producing them has been extensively reviewed. It is difficult to get a high yield of grids by many of these methods, especially if virtually all of the windows must be covered with a tightly bonded, quality film of predictable thickness. We report here a method for producing carbon foils designed to maximize these attributes: 1) coverage of virtually all grid windows, 2) freedom from holes, wrinkles or folds, 3) good adhesion between film and grid, 4) uniformity of film and low noise structure, 5) predictability of film thickness, and 6) reproducibility.Our method utilizes vacuum evaporation of carbon from a fiber onto celloidin film and grid bars, adhesion of the film complex to the grid by carbon-carbon contact, and removal of the celloidin by acetone dissolution. Materials must be of high purity, and cleanliness must be rigorously maintained.

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Effects of high pressure on the crystallization of carbon fiber reinforced polyetheretherketone (CF/PEEK) laminate composites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100177519 ◽

1990 ◽

Vol 48 (4) ◽

pp. 876-877

Author(s):

Hong-Ming Lin ◽

C. H. Liu ◽

R. F. Lee

Keyword(s):

High Pressure ◽

Carbon Fiber ◽

Sample Surface ◽

High Yield ◽

Fiber Reinforced ◽

Laminate Composites ◽

Peek Composite ◽

Continuous Carbon Fiber ◽

Carbon Fiber Reinforced

Polyetheretherketone (PEEK) is a crystallizable thermoplastic used as composite matrix materials in application which requires high yield stress, high toughness, long term high temperature service, and resistance to solvent and radiation. There have been several reports on the crystallization behavior of neat PEEK and of CF/PEEK composite. Other reports discussed the effects of crystallization on the mechanical properties of PEEK and CF/PEEK composites. However, these reports were all concerned with the crystallization or melting processes at or close to atmospheric pressure. Thus, the effects of high pressure on the crystallization of CF/PEEK will be examined in this study.The continuous carbon fiber reinforced PEEK (CF/PEEK) laminate composite with 68 wt.% of fibers was obtained from Imperial Chemical Industry (ICI). For the high pressure experiments, HIP was used to keep these samples under 1000, 1500 or 2000 atm. Then the samples were slowly cooled from 420 °C to 60 °C in the cooling rate about 1 - 2 degree per minute to induce high pressure crystallization. After the high pressure treatment, the samples were scanned in regular DSC to study the crystallinity and the melting temperature. Following the regular polishing, etching, and gold coating of the sample surface, the scanning electron microscope (SEM) was used to image the microstructure of the crystals. Also the samples about 25mmx5mmx3mm were prepared for the 3-point bending tests.

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Catalytic enantioselective synthesis of indolizino[8,7-b]indole alkaloid derivatives based on the tandem reaction of tertiary enamides

Organic Chemistry Frontiers ◽

10.1039/d0qo01147a ◽

2021 ◽

Author(s):

Xin-Ming Xu ◽

Ming Xie ◽

Jiazhu Li ◽

Mei-Xiang Wang

Keyword(s):

Indole Alkaloid ◽

Enantioselective Synthesis ◽

High Yield ◽

Tandem Reaction ◽

Indole Derivatives

An exquisite Pybox/Cu(OTf)2-catalyzed asymmetric tandem reaction of tertiary enamides was developed, which enabled the expeditious synthesis of indolizino[8,7-b]indole derivatives in high yield, excellent enantioselectivity and diastereoselectivity.

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