Fluorophore-Based Mitochondrial Ca2+ Uptake Assay.

The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.

the low, micromolar affinity of MCU and the large amount of cytosolic calcium mitochondria can sequester, MCU-mediated calcium uptake also plays a critical role in clearing transient increases in cytoplasmic calcium and in turn, shapes cellular signaling pathways that use calcium as a secondary messenger (Wheeler et al., 2012). Three, modulations of mitochondrial calcium play an important role in the regulation of apoptosis (Zoratti and Szabo, 1995). Steep increases in mitochondrial calcium levels initiate cell death by inducing the opening of the mitochondrial permeability transition pore in the inner membrane, an event that dissipates the inner mitochondrial membrane potential and releases cytochrome C, Diablo/Smac, and Caspase enzymes from the intermembrane space (Zoratti and Szabo, 1995;Pacher and Hajnoczky, 2001). While these phenomena have been well-characterized, the genetic identity of MCU has only recently been observed, and with that discovery has come the demand for a speedy and reliable way of observing mitochondrial calcium flux.
Our fluorophore-based mitochondrial calcium uptake assay is easy to set up and provides several advantages over other popular methods. HEK-293 cells with activated mitochondria are suspended in a recording buffer along with a membrane-impermeable calcium-sensitive fluorophore. The plasma membrane is permeated with a detergent while leaving the mitochondrial inner membrane in-tact, bringing the mitochondria in direct contact with the buffer. Following this, calcium is added to the cellsuspension and MCU-mediated calcium flux can be followed by observing the changing fluorescence of the fluorophore, which cannot follow calcium into the mitochondria. The highly specific MCU inhibitor, 2 www.bio-protocol.org/e2934 Ru360, is finally added to the cell-suspension to show that the observed change in fluorescence (i.e., calcium flux) is mediated by MCU. One of the most attractive features of the protocol is the speed of set-up and acquisition of flux data. Once cells are ready to harvest, calcium-flux data can be obtained in less than ten minutes. Another important quality of the protocol lies in its simplicity, specifically, in the straight-forward way in which the assay reports calcium flux and identifies MCU as the pathway.
Implicating MCU as the sole calcium uptake pathway in this protocol is the observation that no calcium uptake of any kind is observed in cells lacking MCU. One drawback to the protocol is the inability of it to carefully quantify calcium flux, and for this, a calcium-45 uptake protocol is much preferable. In fact, limited quantification of mitochondrial calcium flux using this protocol is possible if one reports only the relative calcium flux, for example, by comparing two fluxes as a ratio of one over the other.
One of the opaquer yet more popular methods of observing MCU activity aims to follow the changes in mitochondrial calcium levels in intact cells whose mitochondria have been pre-loaded with a membrane-permeable calcium sensitive fluorophore. Because the plasma membranes of these cells are intact, intracellular calcium modulation depends on the release of calcium from the other major intracellular calcium sink, the endoplasmic reticulum (ER), which can be triggered by the addition of histamine to the extracellular buffer. Histamine achieves this by activating the phospholipase C/IP3 pathway, which results in the production of IP3 and concomitant activation of the IP3-receptor in the ER membrane, thereby releasing calcium stores from the ER into to cytoplasm. Because of the spatial proximity of the ER to the mitochondria, activation of the IP3 receptor transiently bathes mitochondria with a high dose of free calcium, which is in turn sequestered by the mitochondrial matrix. In this system, intra-mitochondrial calcium levels are monitored by observing changes in fluorescence of the pre-loaded, membrane-permeable, calcium sensitive probes which have presumably migrated to the mitochondrial matrix. Because MCU is the primary way through which calcium traverses the inner-mitochondrial membrane, it is taken for granted that the observed changes in fluorescence are due to the activation of MCU by these local increases in free calcium. It has even been suggested that the degree to which the fluorescence signal changes upon histamine stimulation is directly proportionate to the degree of MCU functionality. The complexity of this experimental design naturally raises doubts about what's being inferred, namely, that the fluorescence changes upon histamine stimulation are a function of MCU functionality alone and not of, for example, the successful localization of the probe to mitochondria, or of the potential changes to any part of the phospholipase C/IP3 pathway, or of changes in proximity of the ER to mitochondria, or of the amount of calcium stored and/or released by the ER in various cell types under various experimental conditions, any of which may explain the observed fluoresce differences between the conditions tested and which may actually have little to do with MCU functionality.
The experimental design described in detail below aims to reduce these sorts of ambiguities and to clearly report mitochondrial calcium flux mediated by MCU.       6. Add CaCl2 to a final concentration of 10 μM (i.e., 2 μl of 10mM stock). The fluorescence signal will go up as calcium binds to CG-5N. If the cells are harboring activated mitochondria containing functional MCU (i.e., WT-HEK cells), a precipitous declination in fluorescence will immediately follow, signifying MCU-mediated calcium uptake (see Figure 4).

Stir bar speed
The speed of the stir bar can be an important but overlooked factor in determining the quality of the fluorescence trace profile. Spinning too fast can result in cell damage (cells will clump together in the cuvette), while spinning too slowly may limit the speed of reagent mixing within the cuvette, which can be seen in the spectrophotometric data primarily as enhanced noise and hyperbolic-like transitions from one steady-state to another upon the addition of a new reagent to the cuvette. In general, we've found that starting the stir bar in its slowest setting first and then slowly ramping up speed is the best way to find the optimal speed leading to intact cells and quick rates of reagent mixing. In our hands, this ideal speed is, qualitatively, on the slow end within the range of possible stir-bar speeds.

Digitonin
We have found that digitonin from Sigma-Aldrich works particularly well for this experiment. Also, make digitonin stock fresh before each experiment, as this reagent tends to crash out of solution between experiments (within an hour).

Cell density
While the number of HEK cells used in an experiment is completely up to the experimenter, we have found the ideal number to be 2.0 x 10 7 , or 1 fully confluent 10 cm dish.

Cell types
We have performed this experiment exclusively with HEK 293 cells, and cannot say how it might work using other cell types.

Western Blot Analysis
Recording Buffer (RB) does not interfere Western Blot analysis. Simply spin the remaining 0.5 ml of cell slurry down, discard sup (RB), and lyse cells with ice cold lysis buffer (we found RIPA buffer works well for this). Lysing cells with about 50 μl RIPA buffer yields protein concentrations in a range appropriate for the comfortable loading of between 10 μg and 50 μg of protein in a 15 μl or 50 μl per well gel. A high-speed spin step at Step A4 is also required after lysis to pellet cell derbies and to keep the lysate from becoming 'goopy' and unmanageable during loading; keep the supernatant. Follow instructions for your lysis buffer of choice (add protease inhibitors, work on ice, etc.) and carefully quantify protein concentration after the high-speed spin with your method of choice. We always run a loading control gel on which we detect Actin (we load 10 μg protein for this blot). Other gels should be run on which to look for the protein of interest (typically MCU or one of its regulatory partners, but this obviously depends on the experiment and what's desirable to detect). In general, expect to spend time optimizing western blots to see high-quality (high signal to noise) data.

A clear difference between the trace profiles of wild-type cells and MCU-knock out cells (cells in
which MCU has been deleted from the genome) is that in the latter, the slope, after the addition of calcium, is nearly zero, while in the former, the slope is clearly negative. The profile's negative slope after calcium addition is therefore indicative of MCU-mediated calcium uptake.