Fluorometric determination of the DNA content of cells cultured in tissue culture plates

Goppelt‐Struebe, Margarete; Golombek, Marina

doi:10.1016/0022-1759(92)90123-b

Cited by 6 publications

(3 citation statements)

References 11 publications

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“…DNA assay. The amount of DNA in the cultures was estimated after cell lysis in 100 µl of deionized water by measuring DABA-DNA fluorescence (DABA is from Sigma) at 420/510 nm (8). Staining with crystal violet.…”

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confidence: 99%

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.

et al. 2000

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Macrophage dysfunction is a likely mechanism underlying common diabetic complications such as increased susceptibility to infection, accelerated atherosclerosis, and disturbed wound healing. There are no available studies on the function of tissue macrophages in diabetes in humans. We have therefore studied peritoneal macrophages from diabetic type 2-like db/db mice. We found that the release of tumor necrosis factor-␣ and interleukin-1␤ from lipopolysaccharide plus interferon-␥-stimulated macrophages and vascular endothelial growth factor from both stimulated and nonstimulated macrophages was significantly reduced in diabetic animals compared with nondiabetic controls. Nitric oxide production from the stimulated db/db macrophages was significantly higher than that in the db/+ cultures, whereas there was no difference in their ability to generate reactive oxygen species. When studied both at light and electron microscopic levels, macrophages in diabetic animals had an altered morphological appearance compared with those of normal controls. We conclude that the function and morphology of the macrophages are disturbed in db/db mice and that this disturbance is related to the mechanisms underlying common inflammatory and degenerative manifestations in diabetes.

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confidence: 99%

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.

et al. 2000

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“…Deposited calcium was measured with a clinical test combination (Boehringer, Mannheim, 5, Germany). For the determination of DNA content, the spectrofluorometric DABA method was used [5,6].…”

Section: Methodsmentioning

confidence: 99%

Model forin vitro investigation of bone mineralization

Ecsedi¹

1994

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An in vitro model was developed for the investigation of bone cell mineralization processes. Out-grown cells of explanted human nasal bone fragments were selected into three lineages and one of them, the osteoblast-enriched line, was partially characterized. A large number of cells of this line stained readily for alkaline phosphatase. They produced prostanoids, PGl2, PGF2 alpha and PGE2, and osteocalcin in the presence of 1,25(OH)2D3. Cells synthesized collagen and mineralized the developed extracellular matrix. This latter line seems to be appropriate as a model system to find effective compounds which increase bone calcification.

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“…For on-line measurement of dopamine and ATP release, the cells were cultured on dishes (35 mm in diameter) and perfused continuously (See below). DNA content in each culture dish was determined with a spectrofluorometer (FP770, Jasco, Tokyo) according to the method described by Goppelt-Struebe and Golombek [8]. All experiments were done when PC12 cells allowed to grow to subconfluence (about 80% of bottom area of culture dish).…”

Section: Methodsmentioning

confidence: 99%

Release of Dopamine and ATP from PC12 Cells Treated with Dexamethasone, Reserpine and Bafilomycin A1.

Kasai

Ohta

Nakazato

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. The amounts and time courses of dopamine and ATP released from perfused PC12 cells were examined using a simultaneous on-line recording system. High KCl (60 mM) caused dopamine and ATP release with similar time courses. The relative amount of dopamine to ATP in the effluent was 9.5. In PC12 cells cultured with dexamethasone, reserpine or bafilomycin A1 for 2 days, these drugs did not affect increases of intracellular Ca 2+ in response to high KCl. Dexamethasone doubled the amount of dopamine release induced by high KCl without changing the amount of ATP release. High KCl failed to cause dopamine release in reserpine-treated cells but evoked ATP release. Bafilomycin A1 decreased both high KCl-induced dopamine and ATP release. The ratio of released ATP to total adenine nucleotides and adenosine in response to high KCl was not changed by treatment with the drugs. These results suggest that dopamine and ATP are simultaneously released from secretory vesicles of PC12 cells, in which they are stored via different pathways. Similar to dopamine uptake into secretory vesicles, the H + -gradient across the vesicular membrane developed by vacuolar ATPase may play an important role in the vesicular uptake of ATP. KEY WORDS: ATP, dopamine, on-line, PC12 cell, V-ATPase.J. Vet. Med. Sci. 63(4): 367-372, 2001 It is well known that catecholamine (CA) is stored together with adenosine 5'-triphosphate (ATP) in secretory vesicles of the adrenal medulla [32] and pheochromocytoma (PC12) cells [31]. In adrenal chromaffin cells, it has been reported that CA is present in secretory vesicles but not in the cytosol [24] and that 75% of ATP in the cells is in secretory vesicles [7]. ATP stored in secretory visicles has been shown to be released on exocytosis [5]. ATP has been reported to evoke CA release from adrenal chromaffin cells [1,15] and PC12 cells [13] and to inhibit Ca 2+ currents in adrenal chromaffin cells [3,23].Continuous and on-line assay methods for CA released from cultured adrenal chromaffin cells [11,17] and perfused adrenal glands [4,18] have been developed using an electrochemical detector. We previously reported a simultaneous on-line assay method for CA and ATP released from cultured porcine adrenal chromaffin cells using an electrochemical detector and luciferin-luciferase, respectively [14]. Using this system, we found that CA and ATP appeared in the effluent with similar time courses in response to various secretagogues including high KCl. In the sympathetic nerves of the guinea-pig vas deferens, however, it has been reported that the ratios of CA and ATP appearing in the effluent greatly change during stimulation, suggesting the presence of heterogeneous secretory vesicles with respect to the concentrations of CA and/or ATP [28] and post-release degradation of ATP by soluble ectonucleotidases [29]. Furthermore, the vesicles isolated from PC12 cells have been reported to contain CA and ATP at various concentration ratios (CA/ATP) ranging from 13 to 29 [31].It is well known that ATP and CA are taken up ...

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Fluorometric determination of the DNA content of cells cultured in tissue culture plates

Cited by 6 publications

References 11 publications

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.

Model forin vitro investigation of bone mineralization

Release of Dopamine and ATP from PC12 Cells Treated with Dexamethasone, Reserpine and Bafilomycin A1.

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