A rapid, flexibel method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein, Na,K-Atpase and K-p-Nitrophenylphosphatase

Loomis, T. C.; Stahl, William L.

doi:10.1016/0020-7101(86)90015-2

Cited by 7 publications

(2 citation statements)

References 10 publications

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“…The cell pellets were resuspended in HBSS and total cell counts were performed in a hemacytometer. Total protein in lavage fluid and serum was measured by a modification of the Lowry assay adapted for microtiter plates (21). Lavage aliquots were concentrated to -10% of original volume by positive pressure filtration under nitrogen at 4°C using a 5,000-mol wt limit membrane (YM-5; Amicon Co., Danvers, MA).…”

Section: Introductionmentioning

confidence: 99%

Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability.

Martin¹,

Pistorese²,

Emil³

et al. 1989

J. Clin. Invest.

306

134

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Leukotriene B4 (LTB4) is a major product of human alveolar macrophages and has potent chemotactic activity for neutrophils (PMN) in vitro. To evaluate the effects of LTB4 in the normal human lung, we instilled LTB4 (5 X 10-7M, 10 ml) into a subsegment of the right middle lobe and 0.9% NaCI (10 ml) into a subsegment of the lingula using a fiberoptic bronchoscope in 12 healthy human volunteers. 4 h later, we performed bronchoalveolar lavage of the same subsegments. Compared with the NaCl instillation, LTB4 caused a large increase in lavage total cells (NaCI = 6.8±1.0 X 106 vs. LTB4 = 26.4±5.0 X 106, P < 0.01), most of which were PMN (NaCl = 12.2±4.6% vs. LTB4 = 55.7±6.0%, P < 0.001). In contrast, there was only a small increase in lavage total protein, and the lavage total protein correlated weakly with lavage total cells and PMN. The production of superoxide anion by the lavage PMN in response to phorbol myristate acetate was similar to that of peripheral blood PMN. The migration of lavage PMN was normal toward the chemotactic peptide FMLP, but reduced toward LTB4 and zymosan-activated human serum. Morphometric analysis using transmission electron microscopy indicated a selective loss of small granules in the lung neutrophils as compared with peripheral blood neutrophils. The data indicate that in the normal human lung, LTB4 can recruit active PMN into the airspaces without causing a significant change in the protein permeability of the epithelial barrier.

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Section: Introductionmentioning

confidence: 99%

Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability.

Martin¹,

Pistorese²,

Emil³

et al. 1989

J. Clin. Invest.

306

134

View full text Add to dashboard Cite

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“…Total proteins were measured by the Lowry method (22) in an assay modified for microtiter plates (23). To measure the release of preformed proteins from the macrophages, aliquots of the supernatants were dissolved in Laemmli sample buffer (24) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in continuous gradients (10-20 or 5-15% acrylamide) with and without ,B-mercaptoethanol as a reducing agent.…”

Section: Methodsmentioning

confidence: 99%

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage.

Martin¹,

Raugi²,

Merritt³

et al. 1987

J. Clin. Invest.

119

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Human alveolar macrophages release chemotactic activity for neutrophils, prodding a role for alveolar macrophages in regulating inflammation in the lung. As alveolar macrophages produce large amounts of leukotriene B4 (LTB4), a chemotactically active lipoxygenase product of arachidonic acid, we investigated the contribution of LTB4 to the total neutrophil chemotactic activity produced by these cells. Normal human alveolar macrophages were recovered by bronchoalveolar lavage from healthy volunteers and incubated either with the calcium ionophore A23187 for 1 h, or with opsonized zymosan particles or latex beads for 3 h. Nordihydroguaretic acid (NDGA), a relatively specific lipoxygenase inhibitor, blocked the release of neutrophil chemotactic activity after all three stimuli in a dose-dependent manner. This correlated with blockade of LTB4 production as measured by high performance liquid chromatography using freshly isolated alveolar macrophages, as well as blockade of I3H]LTB4 production by macrophages prelabeled with [Hjarachidonate. Molecular sieve chromatography using Sephadex G-50 confirmed that essentially all of the chemotactic activity in the stimulated macrophage supernatants co-eluted with authentic I3HILTB4, and that NDGA completely blocked the chemotactic activity in the eluting fractions. Readdition of authentic LTB4 (1 X iO-7 M) to the NDGA-blocked macrophage supernatants restored the chemotactic activity in the supernatants. The macrophage supernatants did not contain platelet-activating factor-like activity, as measured by the stimulation of V3Hlserotonin release from rabbit platelets, and by high performance liquid chromatography. NDGA did not change the protein-secretion profiles of fresh alveolar macrophages, or of macrophages prelabeled with 135Simethionine. The complement (C) components C5ad.,.z were not detected in any of the supernatants by radioimmunoassay. Concentration of the supernatants by positive pressure filtration (5,000-D membrane) did not augment chemotactic activity in the stimulated supernatants or uncover chemotactic activity in the NDGA-blocked supernatants. As with the 3-h studies, when alveolar macrophages were incubated overnight with opsonized zymosan, all of the increase in chemotactic activity could also be blocked by NDGA. These data indicate that LTB4 is the predominant neutrophil chemotactic factor secreted by the normal resident human alveolar

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A reliable and inexpensive microprocessor system for collection and transfer of raw immunoassay data

Bennett

Sadler

1987

International Journal of Bio-Medical Computing

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A rapid, flexibel method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein, Na,K-Atpase and K-p-Nitrophenylphosphatase

Cited by 7 publications

References 10 publications

Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability.

Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability.

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage.

A reliable and inexpensive microprocessor system for collection and transfer of raw immunoassay data

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