Not surprisingly, considering the importance of calcium, an array of techniques have been and continue to be developed to measure calcium levels and calcium fluxes within cells.
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Choosing the appropriate probe to detect and measure calcium levels for the particular cell type of interest is crucial but can be problematic. In 1928 (Pollack) reported using the dye alizarin sulfonate in ameba which would precipitate calcium ions as purple crystals that could be seen under the microscope. It was later determined that alizarin sulfonate was not specific for calcium and thus not a reliable dye (Kramer, 2015). An alternative to alizarin sulfonate was reported in the 1960s. Aequorin, extracted from jellyfish, is a light emitting protein activated by calcium or strontium (Shimomura and Johnson, 1969). Due to its size however it could not cross membranes, and was limited to large cells were it could be microinjected (Kramer, 2015). Although the issues regarding the size were eventually overcome, the process required to use aequorin is lengthy and susceptible to error due to contamination with calcium found in buffers or even glassware and thus not practical (Borle and Snowdowne, 1986).
It took approximately 50 years from the initial reports of Pollack in 1928 to develop practical calcium indicators that could be used in mammalian cells. The calcium-sensitive fluorescent dye, Quin2, was reported by Tsien, Pozzan and Rink in (1982), this was followed with improved dyes such as Fura-2 and indo-1 several years later (Grynkiewicz, Poenie and Tsien, 1985). The rationale behind the newly developed calcium indicators was based on hybridising a calcium chelator such as EGTA or BAPTA with a fluorescent chromophore (Meldolesi, 2004; Grienberger and Konnerth, 2012). The fluorescent dyes are synthesised in such a way that makes them only temporarily membrane permeable. This is achieved by coupling the chelators to an acetoxymethyl (AM) ester derivative, which is cleaved by endogenous esterases trapping the chelators within the cell (Tsien, 1981).
Since the initial synthetic calcium indicators emerged a variety of fluorescent dyes have become available (Grienberger and Konnerth, 2012). Dyes can come in three forms salts, dextran conjugates or AM esters (Paredes et al., 2009). The salt indicators cannot cross the cell membrane and thus require various loading techniques such as microinjection or lipotransfer using liposomes. Once across the membrane however, they are ready to be used for calcium measurement within minutes albeit for a short period of time, thirty minutes to one hour, due to compartmentalisation of the dye away from the cytosol (Paredes et al., 2009). Dextran conjugates resolve the compartmentalisation issue, however, still require the invasive loading techniques to cross the membrane into the cell (Kreitzer et al., 2000; Paredes
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therefore resolving the cell loading issues equated with the other forms, however compartmentalisation of these dyes is an important drawback (Silver, Whitaker and Bolsover, 1992; Paredes et al., 2009; Kramer, 2015).
Following his success developing fluorescent calcium indicators, Roger Tsein among others in the 1990s, went on to introduce genetically modified proteins that bind calcium sensors (Miyawaki et al., 1997; Persechini, Lynch and Romoser, 1997; Grienberger and Konnerth, 2012). Progress in this field has led to the development of recombinant proteins that can measure very sensitive (nanoscale) calcium fluctuations within a cell (Whitaker, 2010; Looger and Griesbeck, 2012; Kramer, 2015). In fact recently Pandey et al. (2016) carried out calcium measurement experiments with P. falciparum parasites transfected with a recombinant calcium sensing protein. The calcium sensitive indicator yellow cameleon-Nano (YC-Nano) was fused to calmodulin and yellow fluorescent protein was fused to M13 peptide. The M13 peptide is a 27-residue peptide that displays properties of a calmodulin-binding domain. It was derived from skeletal muscle myosin light chain kinase. According to Pandey et al. (2016) in the presence of calcium, calmodulin binds to the M13 peptide which shortens the distance between the two fluorescent proteins, thus increasing the amount of fluorescence.
Once a suitable calcium indicator is identified, a variety of techniques can be developed to study calcium fluctuations and buffering within a cell. Compounds that interfere with calcium homeostasis within a cell are used frequently in research to either raise or reduce the overall calcium levels within a cell or to inhibit or activate specific channels. Examples of these include the calcium chelator EDTA, the calcium ionophore A23187 that raises intracellular calcium, CCBs such as verapamil, calcium channel activators such as Bay K8644 and FPL 64176, and riluzole, an NMDAR inhibitor (Kamal et al., 2015).
5.1.3 Aims
The aim of the work reported in this chapter was to develop a flow cytometry-based method that allowed detection and quantification of fluctuations in calcium within P. falciparum infected cells using a combination of calcium and DNA binding fluorescent dyes. Initially a series of optimisation experiments were carried out to develop a Percoll gradient separation method on infected RBCs which results in obtaining concentrated parasitized cultures. The
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Percoll separated cultures were used in the initial fluorescence tests to determine positive identification of infected cells. Once the use of the dyes was fully optimised a sequence of experiments using different calcium interfering compounds was carried out to determine if calcium fluctuations could be detected.