Fluorescent Ca²⁺ Indicator Conjugates—Section 19.4

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 both low-affinity and high-affinity 10,000 MW dextran conjugates of the fluo-4 indicator (F14240, F36250) and low-affinity and high-affinity 10,000 MW dextran conjugates of our rhod indicator (R34677, 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 19.55). Spectra of the Oregon Green 488 BAPTA-1 dextrans and fluo-4 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 19.58). To solve this problem, we have synthesized fluo-4 dextran (F14240) with a lower Ca²⁺ binding affinity (a batch-dependent K_d for Ca²⁺ ~3 µM). Developed in collaboration with Wade Regehr's laboratory at Harvard University, the low-affinity fluo-4 dextran is a valuable tool for recording Ca²⁺ transients in presynaptic terminals of long axonal projections in heterogeneous fiber tracts. We also offer a high-affinity fluo-4 dextran (K_d for Ca²⁺ ~600 nM, F36250) and high-affinity rhod dextran (K_d for Ca²⁺ ~780 nM, R34676). Coinjection of fluo-4 dextran together with a reference marker (e.g., 10,000 MW Texas Red dextran, D1828; Fluorescent and Biotinylated Dextrans—Section 14.5) may be necessary for initial identification of labeled cells due to the intrinsically weak fluorescence of the fluo-4 indicator in the absence of Ca²⁺.

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 19.55).

Figure 19.58 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 the low-affinity version of fluo-4 dextran (F14240) (lower panel). Each trace represents an average of five recordings. Adapted with permission from Neuron (2000) 27:25.