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.
A light-dependent increase in free Ca2+ concentration in the salamander rod outer segment.
Authors:Matthews HR, Fain GL
Journal:J Physiol
PubMed ID:11306652
'1. The Ca(2+) indicator dye fluo-5F was excited by an argon ion laser to measure changes in free Ca(2+) concentration ([Ca2+]i) in the outer segments of isolated salamander rods rapidly exposed to a 0 Ca(2+), 0 Na(+) solution designed to minimise surface membrane Ca(2+) fluxes. Over 30-60 s of laser ... More
Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.
'BACKGROUND: Microbial biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations ... More
Dopaminergic regulation of dendritic calcium: fast multisite calcium imaging.
Authors:Zhou WL, Oikonomou KD, Short SM, Antic SD,
Journal:Methods Mol Biol
PubMed ID:23296782
'Optimal dopamine tone is required for the normal cortical function; however it is still unclear how cortical-dopamine-release affects information processing in individual cortical neurons. Thousands of glutamatergic inputs impinge onto elaborate dendritic trees of neocortical pyramidal neurons. In the process of ensuing synaptic integration (information processing), a variety of calcium ... More
The effect of light on outer segment calcium in salamander rods.
Authors:Matthews HR, Fain GL
Journal:J Physiol
PubMed ID:12949220
'Calcium acts as a second messenger in vertebrate rods, regulating the recovery phase of the light response and modulating sensitivity during light-adaptation. Since light not only decreases the outer segment calcium concentration ([Ca2+]i) by closing cyclic nucleotide-gated channels but can also increase [Ca2+]i by releasing Ca2+ from buffer sites or ... More
Imaging calcium entry sites and ribbon structures in two presynaptic cells.
Authors:Zenisek D, Davila V, Wan L, Almers W
Journal:J Neurosci
PubMed ID:12684438
'We investigated the location of calcium entry sites and synaptic ribbons in the type-Mb goldfish bipolar neuron and the bullfrog saccular hair cell. Cells were loaded with a fast calcium indicator (Fluo-3 or Fluo-5F) and an excess of a high-affinity but slow Ca buffer (EGTA). The cell surface was imaged ... More