I am doing calcium flux imaging with your Fura-2 calibration kit, but am seeing a large variability in ratio in different places around the slide. I am correcting for uniform illumination, using the product as directed, and sealing the coverslip with nail polish.
The nail polish may be the problem. The Kd value (calcium sensitivity) changes depending upon the dye's environment. Nail polish has solvents that can leech under the coverslip and cause variability. We recommend either going without a sealing or sealing with melted paraffin painted on the coverslip edges with a cotton-tipped applicator (paraffin is hydrophobic and has no solvents).
I need to label cells with Fluo-4, AM, for a calcium flux assay. How long after labeling will the dye be retained?
After loading dye into the cells, intracellular esterases remove the 'AM' moiety from the dye. When the 'AM' group is removed, the dye is able to bind calcium and fluoresce. Since the dye is not covalently bound to any cellular components, it may be actively effluxed from the cell. The rate of efflux is dependent upon the inherent properties of the cell, culture conditions and other factors. The dye may be retained for hours, days or even weeks or lost in a matter of minutes. The use of Probenecid (Cat. No. P36400) limits loss by active efflux.
Mechanism of collagen activation in human platelets.
Authors:Roberts DE, McNicol A, Bose R
Journal:J Biol Chem
PubMed ID:14981087
The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this ... More
The growth-related gene product beta induces sphingomyelin hydrolysis and activation of c-Jun N-terminal kinase in rat cerebellar granule neurones.
Authors:Limatola C, Mileo AM, Giovannelli A, Vacca F, Ciotti MT, Mercanti D, Santoni A, Eusebi F
Journal:J Biol Chem
PubMed ID:10593952
'The growth-related gene product beta (GRObeta) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GRObeta receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GRObeta receptor CXCR2. We also show that, in addition to the known ... More
Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons.
Authors:Fiacco TA, McCarthy KD
Journal:J Neurosci
PubMed ID:14736858
'Spontaneous neurotransmitter release and activation of group I metabotropic glutamate receptors (mGluRs) each play a role in the plasticity of neuronal synapses. Astrocytes may contribute to short- and long-term synaptic changes by signaling to neurons via these processes. Spontaneous whole-cell AMPA receptor (AMPAR) currents were recorded in CA1 pyramidal cells ... More
Control of IP(3)-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin.
Authors:John LM, Mosquera-Caro M, Camacho P, Lechleiter JD
Journal:J Physiol
PubMed ID:11507154
'1. Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP(3)Rs) at low concentrations of IP(3). Ca(2+) puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP(3) into the cell. However, cells appear to have sufficient ... More
Inositol 1,4,5-trisphosphate and ryanodine receptor distributions and patterns of acetylcholine- and caffeine-induced calcium release in cultured mouse hippocampal neurons.
Authors:Seymour-Laurent KJ, Barish ME
Journal:J Neurosci
PubMed ID:7722617
'The distributions of inositol 1,4,5-trisphosphate and ryanodine receptors (InsP3R and RyR) and the patterns of increase in intracellular calcium ion concentration ([Ca2+]i) elicited by their activation were compared in cultured hippocampal neurons. InsP3R and RyR were labeled using specific antibodies and formed small aggregations in the somata and dendrites of ... More