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Ulf T Brunk - One of the best experts on this subject based on the ideXlab platform.
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does the Calcein am method assay the total cellular labile iron pool or only a fraction of it
Biochemical Journal, 2007Co-Authors: Margarita Tenopoulou, Tino Kurz, Paschalisthomas Doulias, Dimitrios Galaris, Ulf T BrunkAbstract:The Calcein-AM (Calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, Calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some Calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting Calcein does not significantly chelate iron at
Calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron. -
Does the Calcein-AM method assay the total cellular ‘labile iron pool‘ or only a fraction of it?
Biochemical Journal, 2007Co-Authors: Margarita Tenopoulou, Tino Kurz, Paschalisthomas Doulias, Dimitrios Galaris, Ulf T BrunkAbstract:The Calcein-AM (acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, Calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular redox-active labile, low mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some Calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, resulting Calcein does not significantly chelate iron at pH
Ayache Bouakaz - One of the best experts on this subject based on the ideXlab platform.
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focused ultrasound influence on Calcein loaded thermosensitive stealth liposomes
International Journal of Hyperthermia, 2015Co-Authors: Anthony Novell, Chantal Al Sabbagh, Jeanmichel Escoffre, Cedric Gaillard, Nicolas Tsapis, Elias Fattal, Ayache BouakazAbstract:AbstractFocused ultrasound (FUS) is a versatile technology for non-invasive thermal therapies in oncology. Indeed, this technology has great potential for local heat-mediated drug delivery from thermosensitive liposomes (TSLs), thus improving therapeutic efficacy and reducing toxicity profiles. In the present study we evaluated the influence of FUS parameters on the release of Calcein from TSLs used to model a hydrophilic drug. Quantitative Calcein release from TSLs (DPPC/CHOL/DSPE-PEG2000: 90/5/5) and non-thermosensitive liposomes (NTSLs) (DPPC/CHOL/DSPE-PEG2000: 65/30/5) was measured by spectrofluorimetry after both water bath and FUS-induced in vitro heating. The heating of TSLs at 42 °C in a water bath resulted in a maximum Calcein release of 45%. No additional Calcein release was observed at temperatures above 42 °C. A similar percentage of Calcein release was achieved when TSLs were exposed to 1 MHz sinusoidal waves at peak negative pressure of 1.5 MPa, 40% duty cycle, for 10 min (i.e. above 42 °C)....
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Focused ultrasound influence on Calcein-loaded thermosensitive stealth liposomes
International Journal of Hyperthermia, 2015Co-Authors: Anthony Novell, Jeanmichel Escoffre, Cedric Gaillard, Nicolas Tsapis, Elias Fattal, Chantal Al Sabbagh, Ayache BouakazAbstract:Focused ultrasound (FUS) is a versatile technology for non-invasive thermal therapies in oncology. Indeed, this technology has great potential for local heat-mediated drug delivery from thermosensitive liposomes (TSLs), thus improving therapeutic efficacy and reducing toxicity profiles. In the present study we evaluated the influence of FUS parameters on the release of Calcein from TSLs used to model a hydrophilic drug. Quantitative Calcein release from TSLs (DPPC/CHOL/DSPE-PEG2000: 90/5/5) and non-thermosensitive liposomes (NTSLs) (DPPC/CHOL/DSPE-PEG2000: 65/30/5) was measured by spectrofluorimetry after both water bath and FUS-induced in vitro heating. The heating of TSLs at 42 degrees C in a water bath resulted in a maximum Calcein release of 45%. No additional Calcein release was observed at temperatures above 42 degrees C. A similar percentage of Calcein release was achieved when TSLs were exposed to 1 MHz sinusoidal waves at peak negative pressure of 1.5 MPa, 40% duty cycle, for 10 min (i.e. above 42 degrees C). No release was detected when NTSLs were heated in a water bath. For both TSLs and NTSLs, the Calcein release was increased by more than 10% for acoustic pressures ranging from 1.5 MPa to 2 MPa. This additional release was attributed to the mechanical stress generated by FUS, which was sufficient to disrupt the liposomal membrane. Furthermore, analysis of cryo-TEM images showed a significant decrease in liposome size (14%) induced by the thermal effect, whereas the liposome diameter remained unaffected by the FUS-triggered non-thermal effects.
Margarita Tenopoulou - One of the best experts on this subject based on the ideXlab platform.
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does the Calcein am method assay the total cellular labile iron pool or only a fraction of it
Biochemical Journal, 2007Co-Authors: Margarita Tenopoulou, Tino Kurz, Paschalisthomas Doulias, Dimitrios Galaris, Ulf T BrunkAbstract:The Calcein-AM (Calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, Calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some Calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting Calcein does not significantly chelate iron at
Calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron. -
Does the Calcein-AM method assay the total cellular ‘labile iron pool‘ or only a fraction of it?
Biochemical Journal, 2007Co-Authors: Margarita Tenopoulou, Tino Kurz, Paschalisthomas Doulias, Dimitrios Galaris, Ulf T BrunkAbstract:The Calcein-AM (acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, Calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular redox-active labile, low mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some Calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, resulting Calcein does not significantly chelate iron at pH
Michel Linder - One of the best experts on this subject based on the ideXlab platform.
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Calcein release behavior from liposomal bilayer influence of physicochemical mechanical structural properties of lipids
Biochimie, 2013Co-Authors: Behnoush Maherani, Azadeh Kheirolomoom, David Geny, Elmira Arabtehrany, Michel LinderAbstract:The design of the drug delivery depends upon different parameters. One of the most noticeable factors in design of the drug delivery is drug-release profile which determines the site of action, the concentration of the drug at the time of administration, the period of time that the drug must remain at a therapeutic concentration. To get a better understanding of drug release, large unilamellar liposomes containing Calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; Calcein was chosen as a model of hydrophilic drug. The Calcein permeability across liposomal membrane (with different compositions) was evaluated on the basis of the first-order kinetic by spectrofluorometer. Also, the effects of liposome composition/fluidity as well as the incubation temperature/pH were investigated. Furthermore, we simulated the digestion condition in the gastrointestinal tract in humans, to mimic human gastro-duodenal digestion to monitor Calcein release during the course of the digestion process. In vitro digestion model ''pH stat'' was used to systematically examine the influence of pH/enzyme on phospholipid liposomes digestion under simulated gastro-duodenal digestion. The results revealed that Calcein permeates across liposomal membrane without membrane disruption. The release rate of Calcein from the liposomes depends on the number and fluidity of bilayers and its mechanical/physical properties such as permeability, bending elasticity. Chemo-structural properties of drugs like as partition coefficient (Log P), H-bonding, polar surface area (PSA) are also determinative parameter in release behavior. Finally, stimulated emission depletion (STED) microscopy was used to study Calcein translocation through liposomal bilayers.
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Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids
Biochimie, 2013Co-Authors: Behnoush Maherani, Azadeh Kheirolomoom, Elmira Arab-tehrany, David Geny, Michel LinderAbstract:The design of the drug delivery depends upon different parameters. One of the most noticeable factors in design of the drug delivery is drug-release profile which determines the site of action, the concentration of the drug at the time of administration, the period of time that the drug must remain at a therapeutic concentration. To get a better understanding of drug release, large unilamellar liposomes containing Calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; Calcein was chosen as a model of hydrophilic drug. The Calcein permeability across liposomal membrane (with different compositions) was evaluated on the basis of the first-order kinetic by spectrofluorometer. Also, the effects of liposome composition/fluidity as well as the incubation temperature/pH were investigated. Furthermore, we simulated the digestion condition in the gastrointestinal tract in humans, to mimic human gastro-duodenal digestion to monitor Calcein release during the course of the digestion process. In vitro digestion model ``pH stat'' was used to systematically examine the influence of pH/enzyme on phospholipid liposomes digestion under simulated gastro-duodenal digestion. The results revealed that Calcein permeates across liposomal membrane without membrane disruption. The release rate of Calcein from the liposomes depends on the number and fluidity of bilayers and its mechanical/physical properties such as permeability, bending elasticity. Chemo-structural properties of drugs like as partition coefficient (Log P), H-bonding, polar surface area (PSA) are also determinative parameter in release behavior. Finally, stimulated emission depletion (STED) microscopy was used to study Calcein translocation through liposomal bilayers.
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Vibrational, calorimetric, and molecular conformational study on Calcein interaction with model lipid membrane
Journal of Nanoparticle Research, 2013Co-Authors: Behnoush Maherani, Azadeh Kheirolomoom, Ewa Rogalska, Beata Korchowiec, Elmira Arab-tehrany, Michel LinderAbstract:Nanoliposomes are commonly used as a carrier in controlled release drug delivery systems. Controlled release formulations can be used to reduce the amount of drug necessary to cause the same therapeutic effect in patients. One of the most noticeable factors in release profiles is the strength of the drug-carrier interaction. To adjust the pharmacokinetic and pharmacodynamic properties of therapeutic agents, it is necessary to optimize the drug-carrier interaction. To get a better understanding of this interaction, large unilamellar liposomes containing Calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; Calcein was chosen as a model polar molecule of biological interest. The thermodynamic changes induced by Calcein and its location in lipid bilayers were determined by differential scanning calorimetry and Raman spectroscopy, respectively. The results reveal that Calcein has no significant influence on thermotropic properties of the lipid membrane, but causing the abolition of pre-transition. The decreasing of the pre-transition can be ascribed to the presence of Calcein near the hydrophilic cooperative zone of the bilayer. The change in intensity of the Raman peaks represents the interaction of Calcein with choline head groups. Moreover, the impact of Calcein on phosphoglyceride Langmuir layers spread at the air-water interface was studied using surface pressure-area and surface potential-area isotherms, as well as polarization-modulation infrared reflection-absorption spectroscopy and Brewster angle microscopy. The results obtained indicate that Calcein introduce no major modification on the systems prepared with pure lipids.
Amy B Harkins - One of the best experts on this subject based on the ideXlab platform.
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light activation of Calcein inhibits vesicle release of catecholamines
ACS Chemical Neuroscience, 2017Co-Authors: Brooke A Miller, Jason B Papke, Vytas P Bindokas, Amy B HarkinsAbstract:Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that Calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when Calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of Calcein. We hypothesized that Calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when Calcein is protected from light during experimentation, little to no reduction of...
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Calcein inhibits vesicle release
Biophysical Journal, 2012Co-Authors: Brooke A Miller, Vytas P Bindokas, Amy B HarkinsAbstract:Charged fluorescent dyes are frequently used as fluid phase makers to detect vesicle fusion events with the cell membrane by means of transient omega-like indentations. We used Calcein (300 μM) to image vesicle fusion from nerve growth factor (NGF)-treated PC12 cells, a model secretory cell line. We stimulated the cells with either high K+ saline or 100 μM nicotine to evoke calcium-dependent vesicle secretion. However, when the cells were stimulated in the presence of Calcein, they showed a reduction in the number of vesicles that were released and/or endocytosed compared to cells that were stimulated in the absence of Calcein. This observation from imaging experiments led us to hypothesize that Calcein was inhibiting vesicle release from NGF-treated PC12 cells. In order to understand whether Calcein was having an inhibitory affect on vesicle secretion and/or reuptake of vesicle membranes, we utilized amperometry to analyze vesicle release of catecholamine transmitter content from NGF-treated PC12 cells. Amperometry is an electrochemical detection method used routinely to measure release of vesicle contents from individual cells with a carbon fiber electrode. Analysis of amperometric spikes provides information as to the number of catecholamine molecules released and the kinetics of release. Our results show that Calcein caused a reduction in the number of released amperometric spike events from 106 ± 13 events measured from control nicotine-stimulated cells, to 60 ± 11 events (p<0.05) in the presence of Calcein. Peak amplitude, half-width, quantal content, or kinetics of rising or falling phase were unaffected by Calcein. These data indicate that Calcein has an inhibitory effect on vesicle fusion with the cellular membrane resulting in reduced amperometry events and a subsequent reduction in uptake of the fluid phase marker when used to track vesicles while imaging.