Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Monteiro, Maria do Céu; Sansonetty, Filipe; Gonçalves, Maria José; O’Connor, José-Enrique

doi:10.1002/(sici)1097-0320(19990401)35:4<302::aid-cyto2>3.0.co;2-j

Cited by 57 publications

(26 citation statements)

References 35 publications

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“…Work in the authors' laboratory using the proposed protocol (Monteiro et al, ) agrees with previous studies by other groups (Davies et al, ; Jennings et al, ; Sage et al, ; Salzman and Ware, ). These reports showed a very fast and transient rise in intracellular free Ca 2+ in platelets stimulated with thrombin or ADP, with the time course and intensity of the response being heterogeneous, but dependent on the type and dose of the agonist.…”

Section: Commentarysupporting

confidence: 87%

Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Monteiro¹,

Gonçalves²,

Sansonetty

et al. 2003

CP Cytometry

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Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular free Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. Moreover, in the clinical setting, whole‐blood techniques have obvious advantages to avoid artifactual platelet activation and allow the maintenance of near‐physiological conditions. This unit describes a fast and sensitive flow cytometric procedure using the Ca2+‐sensitive dye fluo‐3 AM and the platelet‐specific antibody CD41‐PE to determine the kinetics of intracellular Ca2+ mobilization in whole‐blood platelets with minimal manipulation of the samples. The technique may be applied to reveal fast and transient increases in cytosolic calcium upon platelet stimulation with the agonists ADP and thrombin. This protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal‐transduction responses to agonists under near‐physiological conditions, and should be broadly applicable to studies of platelet reactivity.

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Section: Commentarysupporting

confidence: 87%

Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Monteiro¹,

Gonçalves²,

Sansonetty

et al. 2003

CP Cytometry

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“…Intracellular Ca 2+ levels were determined with the Ca 2+ -sensitive fluorochrome fluo-3 AM using flow cytometry as previously described [16]. Briefly, washed human platelets (3 × 10 8 platelets/mL) were loaded with 8 μ M fluo-3 AM for 30 minutes at 37°C in the dark.…”

Section: Methodsmentioning

confidence: 99%

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

Chang

Lee

Tseng

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed thisin vitrostudy to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3βon platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid.

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“…A noncytotoxic detergent, pluronic F-127 (0.1%), was added to increase solubility of Fluo-3/AM. When the time course of intracellular Ca 21 kinetics was analyzed (Monteiro et al, 1999), 6 mM AlCl 3 and/or 10 mM CaCl 2 were added to cell-resuspending medium, pH 4.0, after loading with Fluo-3/AM. PI fluorescence was measured by FACSCalibur with 488-nm (blue) argon (Becton-Dickinson) in the FL2 channel and dichlorofluorescein and Fluo-3 fluorescence in the FL1 channel.…”

Section: Flow Cytometric Studiesmentioning

confidence: 99%

Programmed Cell Death-Involved Aluminum Toxicity in Yeast Alleviated by Antiapoptotic Members with Decreased Calcium Signals

Zheng

Pan

et al. 2006

Plant Physiology

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The molecular mechanisms of aluminum (Al) toxicity and tolerance in plants have been the focus of ongoing research in the area of stress phytophysiology. Recent studies have described Al-induced apoptosis-like cell death in plant and animal cells. In this study, we show that yeast (Saccharomyces cerevisiae) exposed to low effective concentrations of Al for short times undergoes enhanced cell division in a manner that is dose and cell density dependent. At higher concentrations of Al or longer exposure times, Al induces cell death and growth inhibition. Several apoptotic features appear during Al treatment, including cell shrinkage, vacuolation, chromatin marginalization, nuclear fragmentation, DNA degradation, and DNA strand breaks, as well as concomitant cell aggregation. Yeast strains expressing Ced-9, Bcl-2, and PpBI-1 (a plant Bax inhibitor-1 isolated from Phyllostachys praecox), respectively, display more resistance to Al toxicity compared with control cells. Data from flow cytometric studies show these three antiapoptotic members do not affect reactive oxygen species levels, but decrease calcium ion (Ca 21 ) signals in response to Al stress, although both intracellular reactive oxygen species and Ca 21 levels were increased. The data presented suggest that manipulation of the negative regulation process of programmed cell death may provide a novel mechanism for conferring Al tolerance.

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Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Cited by 57 publications

References 35 publications

Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

Programmed Cell Death-Involved Aluminum Toxicity in Yeast Alleviated by Antiapoptotic Members with Decreased Calcium Signals

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