When ion indicators are loaded into cells as their acetoxymethyl (AM) esters, they may translocate to intracellular compartments, where they are still fluorescent but no longer respond to changes in cytosolic ion levels. This problem frequently limits the experiment's duration because sequestration of the indicator into organelles will cause errors in the estimated cytosolic ion levels. Furthermore, Ca2+ indicators such as fura-2 and indo-1 may bind to cellular proteins, which can markedly alter the indicator's response to Ca2+ (Comparison of in vitro and in situ Kd values for various Ca2+ indicators—Table 19.2).
To overcome these limitations, we have prepared dextran conjugates of some Molecular Probes ion indicators (Summary of Molecular Probes fluorescent Ca2+ indicators—Table 19.1). Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. They are also biologically inert due to their uncommon poly-(α-D-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases. Indicator dextrans must be loaded into cells by microinjection, whole-cell patch clamping, microprojectile bombardment or electroporation, or by using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8; ). Calcium indicator dextrans are actively transported in adult nerve fibers over a significant distance and are retained in presynaptic terminals in a form that allows monitoring of presynaptic Ca2+ levels. Dextran conjugates above ~2000 MW are well retained in viable cells, will not readily pass through gap junctions and are less likely to become compartmentalized. Also, fluorescence photobleaching measurements have shown that, as compared with low molecular weight dyes, dextran conjugates are much less likely to bind to proteins. Because dextran conjugates are intrinsically polydisperse and their degree of substitution may vary with the production lot, the Ca2+ dissociation constant of each lot of these indicators should be calibrated independently using one of our Calcium Calibration Buffer Kits (Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).
Fura dextran (F3029) tends to remain in the cytosol without compartmentalization or leakage and is less likely to bind to cellular proteins, making it useful for long-term Ca2+ measurements. Although the spectral response curves of fura dextran are very similar to those of the free dye, its affinity for Ca2+ is somewhat weaker. The dissociation constant for Ca2+ of fura dextran in the absence of Mg2+ varies between 200 nM and 400 nM (measured at ~22°C using our Calcium Calibration Buffers Kits), depending on the molecular weight of the dextran and individual batch characteristics.
Dye compartmentalization has been especially problematic for measurements of ions in plant cells, where the dye is frequently transported out of the cytosol in minutes. Unlike microinjected fura-2 salt, fura dextran is retained for hours in the cytosol of stamen hair cells and Lilium pollen tubes (). A comparison of the dextran conjugates of fura and Calcium Green with conventional indicators for imaging Ca2+ levels in plant and fungal cells has been published.
We offer 3000 MW; 10,000 MW; and 70,000 MW dextran conjugates of the Calcium Green-1 indicator (C6765, C3713, C3714), as well as the 10,000 MW dextran conjugate of the Oregon Green 488 BAPTA-1 indicator (O6798). We have also developed a high-affinity 10,000 MW dextran conjugate of our rhod indicator (R34676). Summary of Molecular Probes fluorescent Ca2+ indicators—Table 19.1 provides a complete list of our Ca2+ indicator dextran conjugates.
A review by Read and co-workers compares the Calcium Green-1 and fura dextrans with conventional indicators for imaging Ca2+ levels in plant and fungal cells. The Calcium Green dextran conjugates (and the other green-fluorescent Ca2+-indicating dextrans) can be coinjected with red-fluorescent Texas Red dextrans (Fluorescent and Biotinylated Dextrans—Section 14.5, Molecular Probes dextran conjugates—Table 14.4) to permit ratiometric measurements of Ca2+ flux in cells that can be microinjected, including oocytes (Figure 19.4.1). Spectra of the Oregon Green 488 BAPTA-1 dextrans match the 488 nm spectral line of the argon-ion laser and standard fluorescein optical filters better than do dextran conjugates of the Calcium Green-1 indicator, which should permit the use of lower probe concentrations to achieve the same signal. The red-orange–fluorescent rhod dextran, which exhibits a 50-fold fluorescence enhancement upon Ca2+ binding, is valuable for multicolor applications and for experiments in cells and tissues that have high levels of autofluorescence.
The visible light–excitable Calcium Green-1 and Oregon Green 488 BAPTA-1 indicator dextrans have been used to:
- Assay Ca2+ influx in taste receptors responding to bitter stimuli
- Detect changes in Ca2+ levels during cell cleavage in mouse and zebrafish embryos
- Follow transmission of changes in cytosolic Ca2+ levels to the nucleus in permeabilized cardiomyocytes and airway epithelial cells
- Label neurons via retrograde or anterograde transport, allowing real-time imaging of neuronal activity ()
- Monitor presynaptic Ca2+ dynamics
- Perform functional imaging of neurons and myocytes during development
- Visualize cADP ribose–induced Ca2+ waves in starfish eggs ()
- Detect depletion of Ca2+ stores in organelles of yeast and mammalian cells
- Examine the regulation of size-specific entry of molecules into the Xenopus oocyte nucleus by the nuclear Ca2+ store
- Monitor changes in intracellular free Ca2+ accompanying photolysis of diazo-2, caged EGTA, caged EDTA or other caged Ca2+ probes
- Perform multiphoton excitation functional imaging of brain activity
In mammalian presynaptic terminals, the response of Calcium Green-1 dextran to successive electrical stimuli progressively weakens due to Ca2+-binding saturation (Figure 19.4.2). In collaboration with Wade Regehr's laboratory at Harvard University, we have synthesized a fluo-4 dextran with a lower Ca2+ binding affinity (a batch-dependent Kd for Ca2+ ~3 µM) for recording Ca2+ transients in presynaptic terminals of long axonal projections in heterogeneous fiber tracts. We also offer a high-affinity rhod dextran (Kd for Ca2+ ~780 nM, R34676).
The lack of a ratiometric Ca2+ response (Loading and Calibration of Intracellular Ion Indicators—Note 19.1) among the visible light–excitable indicator dextrans can be partially circumvented by co-loading Ca2+-insensitive reference markers. Mixtures of Calcium Green dextran and Ca2+-insensitive dextrans (such as the tetramethylrhodamine or Texas Red dextrans in Fluorescent and Biotinylated Dextrans—Section 14.5) have been co-loaded into cells for use in ratio-imaging microscopy (Figure 19.4.1).
Figure 19.4.1 Rhizoid cells from the marine alga Fucus serratus were pressure-microinjected with Ca2+-sensitive 10,000 MW Calcium Green-1 dextran (C3713) and Ca2+-insensitive 10,000 MW Texas Red dextran (D1828). Patterns of Ca2+ elevation following hypoosmotic treatment in dividing cells were visualized by confocal ratio imaging of Calcium Green-1 (excited at 488 nm) and Texas Red (excited at 568 nm). Emission ratio values were pseudocolored to represent calcium concentrations according to the scale bar in the lower left-hand corner. A) Sequential ratio images during the onset of a hypoosmotically induced (100% seawater to 50% seawater) Ca2+ wave shows an initial elevation of Ca2+ in the rhizoid apex, which declines before the onset of Ca2+ elevation arising in the apical nucleus (n1) region. B) A variation in this pattern was evident in a minority of cells where Ca2+ elevations were observed to arise in the subapical nucleus (n2) simultaneously with the apical Ca2+ elevation. The image was contributed by Colin Brownlee, Marine Biological Association of the United Kingdom, Plymouth, UK, and reproduced with permission from Proc Natl Acad Sci U S A (2000) 97:1932.
Figure 19.4.2 Ca2+ transients in rat climbing fiber pre-synaptic terminals evoked by sequences of 10 applied electrical stimulus pulses (20 Hz) monitored by 10,000 MW Calcium Green-1 dextran (C3713) (upper panel) and a low-affinity version of fluo-4 dextran (lower panel). Each trace represents an average of five recordings. Adapted with permission from Neuron (2000) 27:25.
|Low Ca2+||High Ca2+|
|C3713||see Notes||F,D,L||H2O||508||ND||533||H2O||508||ND||533||H2O/Ca2+||260 nM||1, 2, 3, 4, 5, 6, 7|
|C3714||see Notes||F,D,L||H2O||510||ND||535||H2O||510||ND||535||H2O/Ca2+||240 nM||1, 2, 3, 4, 5, 6, 7|
|C6765||see Notes||F,D,L||H2O||510||ND||535||H2O||510||ND||535||H2O/Ca2+||540 nM||1, 2, 3, 4, 5, 6, 7|
|F3029||see Notes||F,D,L||H2O||364||ND||501||H2O||338||ND||494||H2O/Ca2+||240 nM||1, 2, 3, 4, 5, 7|
|O6798||see Notes||F,D,L||H2O||496||ND||524||H2O||497||ND||524||H2O/Ca2+||265 nM||1, 2, 3, 4, 5, 6, 7|
|R34676||see Notes||F,D,L||H2O||549||ND||see Notes||H2O||556||ND||578||H2O/Ca2+||780 nM||1, 2, 3, 4, 5, 7, 8|