Staining by Neutral Red and Trypan Blue in Sequence for Assaying Vital and Nonvital Cultured Cells

DeRenzis, Frank A.; Schechtman, A. M.

doi:10.3109/10520297309116602

Cited by 55 publications

(14 citation statements)

References 3 publications

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“…After 24 h, cells were then re-fed with the same serum-free medium with or without TNF-␣ as indicated. TNF-␣ concentrations up to 100 ng/ml were not cytotoxic to MC3T3-E1 cells as determined by a trypan blue exclusion assay for cell viability (27) and crystal violet analyses for cell layer DNA content (28).…”

Section: Cell Layer Hydroxyproline and Collagen Cross-links-200000mentioning

confidence: 99%

Regulation of Collagen Deposition and Lysyl Oxidase by Tumor Necrosis Factor-α in Osteoblasts

Pischon

Darbois

Palamakumbura

et al. 2004

Journal of Biological Chemistry

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Tumor necrosis factor-␣ (TNF-␣) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-␣ does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-␣ regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-␣ decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-␣ diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is downregulated by TNF-␣ in a concentration-and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-␣ does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts. TNF-␣1 is an inflammatory cytokine produced primarily by monocytes and macrophages and also by a variety of mesenchymal cells. TNF-␣ levels are elevated in various bone disorders such as rheumatoid arthritis, osteoporosis, and periodontitis (1-4). In bone tissue, TNF-␣ inhibits osteoblast function and increases osteoclastogenesis, thus favoring net matrix destruction (5, 6) and the collagenous matrix structure is disrupted by TNF-␣ (7-9). Reports indicate that TNF-␣ somehow inhibits collagen deposition while having little effect on collagen synthesis, although the mechanisms that contribute to this phenomenon have not been elucidated (6, 7).Type I collagen is the major structural protein in the extracellular matrix of bone tissue. Normal collagen structure and the balance between production, deposition, and degradation of collagen are important in the development and maintenance of skeletal tissue (1). Collagen biosynthesis is a multistep process that involves intracellular posttranslational modifications, assembly of procollagen chains, secretion, extracellular processing, and cross-linking to form a mature functional matrix (10). The mechanisms...

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Section: Cell Layer Hydroxyproline and Collagen Cross-links-200000mentioning

confidence: 99%

Regulation of Collagen Deposition and Lysyl Oxidase by Tumor Necrosis Factor-α in Osteoblasts

Pischon

Darbois

Palamakumbura

et al. 2004

Journal of Biological Chemistry

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“…This assay is based on 51Cr-labelling of intact cells and was carried out in KR-HEPES medium containing 10 mg/ml HSA and 3 or 20 mM glucose. Viable cell count was performed with 0.2% (w/v) trypan blue alone [21] or with trypan blue and neutral red used in sequence as described by DeRenzis and Schechtman [2].…”

Section: Viability Testsmentioning

confidence: 99%

The preparation of, and studies on, free cell suspensions from mouse pancreatic islets

1974

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Summary. Pancreatic islets, isolated from the pancreas of obese-hyperglycemic mice, were used to prepare free islet cells in suspension. Batches of 100 islets were disrupted by meeha.nieal shaking for 10 see in a Ca~+-free HEPES-buffered Krebs-Ringer medium containing 1 mM EGTA. From 200--500 islets, about 2.2 • 106 cells could be obtained in suspension, corresponding to a yield of roughly 55% as calculated from the content of DNA in cells and islets. The isolated islet cells appeared wellpreserved in the light and the electron microscope and seemed to exhibit a high degree of viability as judged by viable cell counts, a radioactive assay for lysis and insulin release. Insulin release from islet cell suspensions, as calcuIated per cell, per content of DNA or insulin or per packed cell volume, was stimulated by glucose alone or iu combination with theophylline. The glucose response was low compared with that of intact isolated islets.

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“…which was then excised, minced, and placed in a [ 11] and ranged from 0.5 to 1.0 mg/ml. Cell viabil ity was determined by the trypan blue dye exclusion test [12] and only material in which at least 95% of the cells were viable was used.…”

Section: Methodsmentioning

confidence: 99%

Displacement of Alpha- and Beta-Radioligands by Specific Adrenergic Agonists in Rat Pancreatic Islets

et al. 1983

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Secretion of insulin is increased by β-adrenergic agonists and inhibited by α-adrenergic agonists. However, administration of epinephrine, which acts on both types of receptors, inhibits insulin secretion. A preliminary study using [³H]-dihydroergocryptine and [³H]-dihydroalprenolol as the respective α- and β-receptor binding ligands, surprisingly revealed a preponderance of β-binding sites in normal rat pancreatic islets. The present study, using displacement by epinephrine, norepinephrine, isoproterenol and clonidine validated the use of these radioligands as appropriate for specific receptor binding in pancreatic islet cells. The islets were found to have 55 fmol/mg protein of α-adrenergic receptor sites and 170 fmol/mg protein of β-receptor sites. The affinity of both α- and β-receptors for epinephrine was similar, as judged by the displacement of either radioligand, thus ruling out a preferential affinity of α-receptor binding as an explanation for the α-inhibition of insulin secretion. The data on radioligand displacement by clonidine indicate that the α-receptor is of the α_2-type.

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Staining by Neutral Red and Trypan Blue in Sequence for Assaying Vital and Nonvital Cultured Cells

Cited by 55 publications

References 3 publications

Regulation of Collagen Deposition and Lysyl Oxidase by Tumor Necrosis Factor-α in Osteoblasts

Regulation of Collagen Deposition and Lysyl Oxidase by Tumor Necrosis Factor-α in Osteoblasts

The preparation of, and studies on, free cell suspensions from mouse pancreatic islets

Displacement of Alpha- and Beta-Radioligands by Specific Adrenergic Agonists in Rat Pancreatic Islets

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