Calcium Dye Research Articles

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Fluorescence correlation spectroscopy experiments to quantify free diffusion coefficients in reaction-diffusion systems: The case of Ca^{2+} and its dyes.

Many cell signaling pathways involve the diffusion of messengers that bind and unbind to and from intracellular components. Quantifying their net transport rate under different conditions then requires having separate estimates of their free diffusion coefficient and binding or unbinding rates. In this paper, we show how performing sets of fluorescence correlation spectroscopy (FCS) experiments under different conditions, it is possible to quantify free diffusion coefficients and on and off rates of reaction-diffusion systems. We develop the theory and present a practical implementation for the case of the universal second messenger, calcium (Ca^{2+}) and single-wavelength dyes that increase their fluorescence upon Ca^{2+} binding. We validate the approach with experiments performed in aqueous solutions containing Ca^{2+} and Fluo4 dextran (both in its high and low affinity versions). Performing FCS experiments with tetramethylrhodamine-dextran in Xenopus laevis oocytes, we infer the corresponding free diffusion coefficients in the cytosol of these cells. Our approach can be extended to other physiologically relevant reaction-diffusion systems to quantify biophysical parameters that determine the dynamics of various variables of interest.

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method forlimiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (>24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 °C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at >24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Analysis of the dynamics of temporal relationships of neural activities using optical imaging data

The temporal relationship between the activities of neurons in biological neural systems is critically important for the correct delivery of the functionality of these systems. Fine measurement of temporal relationships of neural activities using micro-electrodes is possible but this approach is very limited due to spatial constraints in the context of physiologically valid settings of neural systems. Optical imaging with voltage-sensitive dyes or calcium dyes can provide data about the activity patterns of many neurons in physiologically valid settings, but the data is relatively noisy. Here we propose a numerical methodology for the analysis of optical neuro-imaging data that allows robust analysis of the dynamics of temporal relationships of neural activities. We provide a detailed description of the methodology and we also assess its robustness. The proposed methodology is applied to analyse the relationship between the activity patterns of PY neurons in the crab stomatogastric ganglion. We show for the first time in a physiologically valid setting that as expected on the basis of earlier results of single neuron recordings exposure to dopamine de-synchronises the activity of these neurons. We also discuss the wider implications and application of the proposed methodology.

ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15min followed by 24h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1μg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70mM K+. A sub effective concentration of ConBr (0.01µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Abstract 207: Electrically-induced Calcium Handling in Human Cardiac Progenitor Cells

The discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs) has raised the question of the mechanisms involved in regenerative properties of this niche of cells. The physiology of human CPCs is not well understood, and a thorough understanding of the Ca 2+ handling ability of these cells is crucial in order to fully realize their regenerative potential. Therefore, to characterize the mechanism of electrically-induced Ca 2+ handling in human CPCs, we have used cells loaded with fluo-4 calcium dye and imaged using confocal microscopy to determine the spatial and temporal profile of cytosolic Ca 2+ upon electrical stimulation. Upon activation of electrical stimulation, a large increase in cytosolic Ca 2+ was observed followed by oscillations of the cytosolic Ca 2+ . The electrically-induced Ca 2+ oscillations were found to be dependent on the activity of L-type Ca 2+ channels (LTCC) and the presence of extracellular Ca 2+ . Furthermore, these oscillations could be abolished by pre-treatment of the cells with the inositol 1,4,5-trisphosphate receptor (IP 3 R) antagonist 2-APB. High-speed confocal imaging of the electrically-induced Ca 2+ oscillations in hCPCs revealed that these cells oscillate in a Ca 2+ wave-like fashion with activity originating in isolated regions of the cell and propagating throughout the cytosol and nucleus. In conclusion, we propose that Ca 2+ -dependent regulatory mechanisms can be exploited to influence their differentiation potential leading to improved function in cardiac regenerative medicine.

Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45–150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6–8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

0291 : Mitochondrial permeability transition pore opening might contribute to ventricular arrhythmia maintenance

Calcium leak from the sarcoplasmic reticulum (SR) favors cardiac sudden death through a calcium overload mechanism. Here we investigate the crosstalk between the calcium overload SR-mediated and the mitochondrial permeability transition pore (mPTP) opening during a ventricular arrhythmic episode. a burst pacing protocol was developed to induce ventricular arrhythmia in the perfused rat heart. Surface electrocardiogram, left ventricular pressure, and oxygen consumption were monitored. Epicardial fluorescence was recorded from either a mitochondrial membrane potential dye or an intracellular calcium dye. ECG signals were used to assess both arrhythmia complexity and duration. Hearts were perfused with tacrolimus (FK506) alone or in combination with cyclosporin A (CsA) to promote SR calcium leak or inhibit mPTP, respectively. Prior to arrhythmia induction, FK506 promoted mitochondrial depolarization and intracellular calcium overload in a CsA-sensitive manner. More than 90% of hearts developed complex ventricular arrhythmias in the FK506 group and this was accompanied by a strong mitochondrial membrane depolarization. In contrast, in the FK506 + CsA group, 100% of the hearts spontaneously defibrillate after a few seconds of arrhythmias. In this group, following arrhythmia induction, mitochondrial depolarization amplitude, intracellular calcium overload and heart efficiency depression was decreased in comparison to the FK506 group. Here we suggest that the FK506-induced calcium overload promotes mitochondrial calcium accumulation and leads to mPTP opening. We propose that mPTP opening should be considered as an arrhythmogenic substrate. The author hereby declares no conflict of interest

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BackgroundMutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca2+ release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca2+ handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n′,n′-tetraacetic acid (EGTA) to measure Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions.MethodsStandard and confocal fluorescence microscopy was used to study Ca2+ release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student’s test with significance set at p < 0.05.ResultsWe found that the peak value of Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites.ConclusionsOverall, these results suggest that decreased Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Real-Time Analysis of Calcium Signals during the Early Phase of T Cell Activation Using a Genetically Encoded Calcium Biosensor

Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux after stimulation with an APC in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naive T cells in the lymph node occurs in distinct phases. In phase I, T cells make multiple transient contacts with dendritic cells before entering a phase II, where they exist in stable clusters with dendritic cells. It has been suggested that T cells signal during transient contacts of phase I, but this has never been shown directly. Because time-dependent loss of calcium dyes from cells hampers long-term imaging of cells in vivo after antigenic stimulation, we generated a knock-in mouse expressing a modified form of the Cameleon fluorescence resonance energy transfer reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro, we observed transient, oscillatory, and sustained calcium flux after contact with APC, but these behaviors were not affected by the type of APC or Ag quantity, but were, however, moderately dependent on Ag quality. In vivo, we found that during phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naive T cells signal during phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of phase II.

Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels.

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.

LFP-guided targeting of a cortical barrel column for in vivo two-photon calcium imaging.

Two-photon microscopy of bulk-loaded functional dyes is an outstanding physiological technique that enables simultaneous functional mapping of hundreds of brain cells in vivo at single-cell resolution. However, precise targeting of a specific cortical location is not easy due to its fine dimensionality. To enable precise targeting, intrinsic-signal optical imaging is often additionally performed. However, the intrinsic-signal optical imaging is not only time-consuming but also ineffective in ensuring precision. Here, we propose an alternative method for precise targeting based on local field potential (LFP) recording, a conventional electrophysiological method. The heart of this method lies in use of the same glass pipette to record LFPs and to eject calcium dye. After confirming the target area by LFP using a glass pipette, the calcium dye is ejected from the same pipette without a time delay or spatial adjustment. As a result, the calcium dye is loaded into the same ensemble of brain cells from which the LFP was obtained. As a validation of the proposed LFP-based method, we targeted and successfully loaded calcium dye into layer 2/3 of a mouse barrel column.

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

Simultaneous Impedance Spectroscopy and Optical Microscopy to Investigate Cells Attachment and Spreading

In a normal environment cells are responding to the signals coming from their external environment, and the way they react often determines their fate. These signals can come from multiple soluble chemicals (soluble cues) such as secreted proteins from neighboring cells. These cues usually trigger incredibly complicated pathway of events inside the cells, which lead to certain cellular responses. The mechanisms behind the responses are even more sophisticated as physical property of surrounding environment (adhesive cues) such as ligands in extracellular matrix (ECM) have also their own effect on the signaling pathways and the overall cells responses. Specifically, the Arg-Gly-Asp ligands present in ECM have been mainly known for determining the extent of the cells attachment and spreading in its environment through binding to cellular integrins. It is crucial to achieve a better understanding of the possible links between signaling networks provoked by soluble and adhesive cues. We have coupled fluorescence microscopy and electrochemical Impedance spectroscopy simultaneously to provide more comprehensive information about this connection. To model the adhesive cues, the interdigitated Indium−tin oxide (ITO) surfaces were modified to present different RGD densities. Fluorescence microscopy was used to monitor the activity of G-protein coupled receptors (GPCR) by measuring the intracellular calcium flow, while the impedance spectroscopy was used to track the minute morphological changes all in response to histamine, as an examples of soluble cues, addition on the modified ITO surfaces. The ITO surfaces with different RGD ligand spacing were prepared by modifying the surfaces with different ratios of hydroxyl-terminated and methoxy-terminated 1-aminohexa(ethylene oxide), followed by incubation in 20 µg/ml RGD solution on a cell-inert background, which was developed on the surface previously. The cell-surface interaction was investigated by seeding 104 HeLa cells/ cm2 on surfaces for 3 h, following by fixing, and staining with phalloidine-Alexa 488 to determine the extent of cells spreading. For impedance spectroscopy, the resistance of the cell layer was measured at 40 kHz, since plating the cells, while the calcium flow in response to addition of histamine was measured using fluorescence microscopy after loading the cells with Fura-2 AM calcium dye. A custom-made chamber from Micrux (Oviedo, Spain) was used to run the simultaneous experiment. The fixed cell-surface study showed that the cells morphology and spreading is highly dependent to the amount of RGD ligands present on surfaces. The microscopy results indicates that histamine mediated GPCRs activity can significantly induce the flow of calcium in cytosol. In contrast, the impedance spectroscopy showed that altering the ECM-cell integrin interaction by introducing different RGD ligands to the surface modulates the cells morphology on the surface. More importantly, we observed that the presence of different RGD ligands on the surfaces dramatically changed the amount of released calcium as Figure 1 shows. Surfaces modified with 1:103 of hydroxyl-terminated and methoxy-terminated 1-aminohexa(ethylene oxide) was demonstrating the highest amount of released intracellular calcium, while it showed the lowest increase in the amount of impedance among the surfaces that were used in this experiment. Cells response to histamine molecules, as soluble cues, trough GPCR was investigated on interdigitated ITO surfaces modified with different RGD ligand densities which acts as cells adhesive cues. The results indicate strong correlation between the effect of soluble cues and adhesive cues. Understanding this highly interconnected signaling networks is vital to biomedical engineering science. Figure 1. The percentage of increase in the ratiometric value of Fura-2 AM in response to 100 µM histamine on ITO surfaces modified with different ratio of 1-aminohexa(ethylene oxide) to 1- aminohexa(ethylene oxide) monomethyl ether. The image is an example of the fluorescence images showing the increase in calcium flow in response to histamine, where blow-red refers to low-high. Figure 1

Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe

BackgroundBiocytin has found numerous uses as a neuronal tracer, since it shows both antero- and retrograde transport in neuronal tracts. The main advantage of biocytin lies in the comprehensive intracellular distribution of the molecule, and in effective detection using avidin-based reactions. The main drawback is that biocytin cannot be visualized in live tissue. New methodWe demonstrate that TMR biocytin, a conjugate of biocytin and a rhodamine fluorophore, is an effective neuronal tracer in live tissue when applied by electroporation. ResultsThe initial fiber transport velocity of TMR biocytin is high—5.4mm/h. TMR biocytin can be used in conjunction with AM calcium dyes to label neuronal somas from distances of several millimetres, and record calcium transients during the course of a few hours. Juxtacellular application of TMR biocytin leads to fast anterograde transport with labeling of local synapses within 10min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue. Comparison with existing methodsRetrograde labeling with TMR biocytin enables long-range neuronal visualization and concurrent calcium imaging after only a few hours, which is substantially faster than other fluorescence-based tracers. The green emitting Atto 488 biotin is also taken up and transported retrogradely, but it is not compatible with standard green emitting calcium dyes. ConclusionsTMR biocytin is an attractive neuronal tracer. It labels neurons fast over long distances, and it can be used in conjunction with calcium dyes to report on neuronal activity in retrogradely labeled live neurons.

Optical electrocorticogram (OECoG) using wide-field calcium imaging reveals the divergence of neuronal and glial activity during acute rodent seizures.

The role of glia in epilepsy has been widely debated. Using in vivo bulk loading of calcium dyes, we imaged neuronal and glial activity in an acute pharmacologic rodent model of neocortical seizures. Optical calcium-based ECoG maps revealed that neuronal waves propagated rapidly and remained mostly confined to the seizure focus. Glial waves were triggered by ictal onset but propagated slowly in a stereotypical fashion far beyond the seizure focus. Although related at their onset, the divergence of these two phenomena during seizure evolution calls into question their interdependence and the criticality of the role of glia in seizure onset and neurovascular coupling. This article is part of a Special Issue entitled "Status Epilepticus".

Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1) using a single calcium dye provides an additional channel for surface marker characterization, 2) allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3) can measure total calcium flux and additionally, the proportion of responding cells, 4) can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX), on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells.

Tendon Cells Demonstrate Store‐Operated Calcium Entry Capacity and Differences in Calcium Signaling Through Aging

Calcium is an important and ubiquitous second messenger, controlling a wide variety of cellular functions in virtually all cells in the mammalian body. In particular, calcium homeostasis is known to be a critical biomarker for tendon cell (tenocyte) behavior, serving as an essential feature of proper tenocyte function and mechano-transduction capability. However, the molecular mechanisms of how intracellular calcium levels are regulated within tenocytes, as well as the changes that these processes undergo through the aging process have remained largely unexplored. Using the ratiometric fluorescent calcium dye Fura-2, we showed for the first time evidence of store-operated calcium entry (SOCE) in primary tenocytes obtained from tail tendon fascicles of the mouse. In addition, since studies have shown that the process of aging yields a predominant decline in tissue and cell health, we further investigated calcium homeostasis in both young (4-6 months) and aged (20-22 months) mice to determine whether or not calcium regulation is altered through the process of aging. Overall, aged tenocytes demonstrated a reduced SOCE response and higher calcium “resting” levels, when compared to young tenocytes. This work shows for the first time, tenocyte capacity for SOCE, as well as differences in this process between younger and older cells, and opens new investigative avenues for tendon biology and potential treatments for tendon disorders.

A human platelet calcium calculator trained by pairwise agonist scanning.

Since platelet intracellular calcium mobilization [Ca(t)]i controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Pairwise Agonist Scanning (PAS) deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50; 154 conditions including a null condition) to stimulate platelet P2Y1/P2Y12 GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t)]i data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t)]i beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.