Cell Biology

The characteristics of Ca²⁺ and H⁺ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H⁺ concentration and increase in cytoplasmic Ca²⁺ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca²⁺ and H⁺ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca²⁺ and H⁺ fluxes across transformed Pichia pastoris cells in response to glycerol and polyethylene glycol 6000 (PEG6000) treatments. The transformed yeast cells are loaded onto a coverslide pre-processed in the poly-L-lysine solution (0.1% w/v aqueous solution). After cell immobilization, microelectrodes are positioned above a monolayer of attached cell population. Micro-volts differences are measured at two excursion points manipulated by a computer. Micro-volts differences could be converted into ion fluxes using the ASET 2.0 and iFluxes 1.0 Software. The method is expected to promote the application of NMT in microbiology. We are very grateful to Younger USA (Xuyue Beijing) NMT Service Center for their critical reading of the manuscript.

Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cell lines in which organic-anion transporters are particularly active (e.g. CHO, HeLa), addition of probenecid, an inhibitor of anion transport, is required for retention of this dye in the cells. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. This protocol can be applied to most mammalian cell lines expressing both endogenous and transiently/stably transfected receptors. The method is sensitive enough to be used for low-expressing systems or high throughput screening of target of interest.
Note: The method does not differentiate the Ca²⁺ mobilization induced by Gqα from the Ca²⁺ mobilization induced by Gβγ.

This is a protocol to analyze the functional response of single CHO-K1 cells to a given treatment in terms of changes in free cytosolic calcium concentration ([Ca2+]i). This is possible by using the Ca2+ indicator dye Fura-2 AM, a polyamino carboxylic acid that binds to free intracellular calcium and is excited at 340 nm and 380 nm. The ratio of the emissions at 505 nm after excitation with those wavelengths is directly correlated to the amount of intracellular calcium. This protocol can be applied to other cell types (cell lines or primary cell cultures) by changing the culture conditions accordingly to the cell type.

This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.