Differences between Cu- and Fe–Cu nanoflowers in their interactions with fluorescent probes ANS and Fura-2 and proteins albumin and thrombin

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.

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A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168578 ◽

1994 ◽

Vol 52 ◽

pp. 168-169

Author(s):

Martin Poenie ◽

Akwasi Minta ◽

Charles Vorndran

Keyword(s):

Metal Binding ◽

Acetoxymethyl Ester ◽

Binding Properties ◽

Fura 2 ◽

New Family ◽

Anion Transporter ◽

Calcium Indicator ◽

Calcium Indicators ◽

High Calcium ◽

Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

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Live Confocal Analysis of Fertilization and Early Development

Microscopy and Microanalysis ◽

10.1017/s1431927600031135 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1012-1013

Author(s):

Uyen Tram ◽

William Sullivan

Keyword(s):

Drosophila Melanogaster ◽

Embryonic Development ◽

Real Time ◽

Early Development ◽

Fluorescent Probes ◽

Primary Defect ◽

Fluorescent Analysis ◽

Dynamic Event ◽

Enormous Amount ◽

Fluorescent Studies

Embryonic development is a dynamic event and is best studied in live animals in real time. Much of our knowledge of the early events of embryogenesis, however, comes from immunofluourescent analysis of fixed embryos. While these studies provide an enormous amount of information about the organization of different structures during development, they can give only a static glimpse of a very dynamic event. More recently real-time fluorescent studies of living embryos have become much more routine and have given new insights to how different structures and organelles (chromosomes, centrosomes, cytoskeleton, etc.) are coordinately regulated. This is in large part due to the development of commercially available fluorescent probes, GFP technology, and newly developed sensitive fluorescent microscopes. For example, live confocal fluorescent analysis proved essential in determining the primary defect in mutations that disrupt early nuclear divisions in Drosophila melanogaster. For organisms in which GPF transgenics is not available, fluorescent probes that label DNA, microtubules, and actin are available for microinjection.

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Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645981 ◽

1987 ◽

Vol 58 (02) ◽

pp. 737-743 ◽

Cited By ~ 12

Author(s):

Frarnçois Lanza ◽

Alain Beretz ◽

Martial Kubina ◽

Jean-Pierre Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fluorescent Indicators ◽

Fura 2 ◽

Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

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Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655971 ◽

1997 ◽

Vol 77 (02) ◽

pp. 376-382 ◽

Cited By ~ 15

Author(s):

Bruce Lages ◽

Harvey J Weiss

Keyword(s):

Platelet Rich Plasma ◽

Limited Range ◽

Dense Granule ◽

Receptor Desensitization ◽

Fura 2 ◽

Storage Pool ◽

Low Levels ◽

The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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