Materials and Reagents

Round glass coverslips 12 mm (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 1014355112NR15 ) - sterile upon autoclave cycle
24-well plate (Corning, Costar^®, catalog number: 3524 )
MDA-MB-231 cell line
MDA-MB-468 cell line
BT-549 cell line
Aequorin-expressing plasmids (Brini, 2008)
Gelatin
Collagen
Coelenterazine 0.5 mM stock solution, in methanol (IS Chemical Technology, catalog number: I14-2266 ) - aliquot in 50 μl aliquots. Store at -80 °C. Save it from light
Agonist (e.g., ATP, Histamine, Bradykinin, Caffeine, Carbachol, Glutamate)
Note: In this protocol example 100 µM ATP is used.
Milli-Q water
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M2670 )
Potassium dihydrogen phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: NIST200B )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: M2773 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 449709 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Digitonin (Sigma-Aldrich, catalog number: D5628 or D141 )
Krebs-Ringer modified buffer (KRB) (see Recipes)
Digitonin lysis solution (see Recipes)

Equipment

Windows-based computer
Perfusion chamber (Elettrofor)
Low noise photomultiplier (Hamamatsu Photonics K. K., model: H7360-01)
Peristaltic pump (Gilson’s MINIPULS^® 3)
Water bath (temperature-controlled)
Photon-counting unit (Hamamatsu Photonics K. K., model: C8855-01 )

Note: Equipment is assembled as depicted in Figure 1.

Figure 1. Equipment. A. Fully equipped aequorinometer is composed of: (1) computer, (2) perfusion chamber, (3) photomultiplier, (4) peristaltic pump, (5) water bath, (6) photon-counting unit. B. During the experiment, the perfusion chamber is placed in close proximity to the photomultiplier, protected from light.

Procedure

Day 1
Plate cells on 12 mm round glass coverslips, in a 24-well plate at 30-50% confluence (about 70,000 cells/well) and let them grow in their specific medium (Figure 2). Pre-treatment of coverslips with gelatin, collagen or poly-lysine to increase cell adherence is not mandatory, but could be recommended for specific cell types.

Figure 2. Different mammalian cell lines. MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cell lines at around 40% confluence.
Day 2
Transfect cells with the proper aequorin-expressing plasmids, according to the expected free [Ca²⁺] present in the subcellular compartment (Brini et al., 1995; Brini, 2008). Choose the best transfection/infection protocol depending on the targeted cell type. In this example protocol, Aequorin-wt and mitochondrial mutated aequorin (Asp119Ala) are used.
Day 3
1. 24 h after transfection remove medium from the cell plate.
2. Wash cells with KRB solution once.
3. Incubate cells at 37 °C for 90 min with 5 µM coelenterazine in 200 µl KRB solution.
4. Transfer a coverslip containing the transfected cells to the perfusion chamber. Fix the perfusion chamber in close proximity to the photomultiplier (2-3 mm distance) (Figure 3).
5. Continuously perfuse cells with KRB, at 37 °C in a water bath. Agonists and other drugs should be added to the same solution.
6. Switch on the photomultiplier and start recording the light emission, ideally with a time span of 1 sec (the output of the amplifier-discriminator is captured by the photon-counting unit connected to a Windows-based computer).
7. As soon as the background values are stable, stimulate cells with an appropriate agonist concentration, in order to induce Ca²⁺ release from the endoplasmic reticulum (ER).
8. Follow light emission until suppression of Ca²⁺ signals.
9. Terminate the experiment by lysing cells with the digitonin solution, thus discharging the remaining aequorin pool.
10. Proceed with data analysis and calibration, as described below.
  
  Figure 3. Experimental procedure. The coverslip coated with transfected cells is moved from the plate (A) to the perfusion chamber (B). The perfusion chamber is then placed near the photomultiplier (C).

Data analysis

In order to convert the luminescence signal in [Ca²⁺], an algorithm has been developed. Three variables are required:

The total amount of luminescence that each sample would emit in presence of saturating [Ca²⁺]. In order to calculate this parameter, at the end of each experiment cells are lysed with a digitonin-containing solution (as reported in the Procedure section), and the luminescence emitted by the residual aequorin is measured. Since the total amount of luminescence is directly dependent on the amount of aequorin, this parameter takes into account differences in transfection efficiency.
L_max, i.e., the luminescence that would be emitted at a certain time point if all the aequorin had been suddenly discharged. Since aequorin is being constantly consumed, L_max progressively decreases during the experiment. At each time point, L_max is calculated by subtracting the luminescence recorded before that point from the total light output of the whole experiment.
L, i.e., the rate of photon emission at any instant during the experiment.
The [Ca²⁺] at each time point is a function of L/L_max (Figure 4). The presence of three Ca²⁺ binding sites in the aequorin molecule is responsible for the steep relationship between photon emission rate and free [Ca²⁺]. The rate of luminescence is independent of [Ca²⁺] at very high (> 10^-4 M) and very low [Ca²⁺] (< 10^-7 M). However, it is possible to expand the range of [Ca²⁺] that can be monitored by choosing the proper recombinant aequorin construct (Figure 5).
Data should be plotted as average of Ca²⁺_max peaks from replicates of each condition. At least 6 replicates per condition are recommended. In addition, a representative Ca²⁺ trace for each group of samples should be shown.

Figure 4. Representative Ca²⁺ calibration curve. At the end of the experiment, after cell lysis, the total amount of aequorin can be estimated and L/L_max can be calculated for each data point, thus allowing the conversion of light emitted into free Ca²⁺ concentration values. N = 6.

Figure 5. Representative traces of intracellular Ca²⁺ measurements. A. Mitochondrial Ca²⁺ uptake upon 100 µM ATP stimulation, using a mutated isoform (Asp119Ala) of aequorin. B. Cytosolic Ca²⁺ transients upon ATP stimulation, using the wild-type aequorin. C. ER Ca²⁺ uptake monitored by adding 1 mM CaCl₂ to a Ca²⁺-free KRB, and subsequent Ca²⁺ release induced by agonist stimulation (ATP).

Notes

Although aequorin luminescence is influenced neither by K⁺ nor Mg²⁺ (which are the most abundant cations in the intracellular environment and thus the most likely source of interference in physiological experiments) both ions are competitive inhibitors of Ca²⁺-activated luminescence. pH also affects aequorin luminescence at values below 7. For these reasons, experiments with aequorin need to be done in well-controlled conditions of pH and ionic concentrations, notably of Mg²⁺.
To stimulate Ca²⁺ release from ER, the appropriate agonist should be selected for each cell type (e.g., ATP, Histamine, Bradykinin, Caffeine, Carbachol, Glutamate). The choice depends on cell-type specific expression of the G protein-coupled plasma membrane receptors.

Recipes

Krebs-Ringer modified buffer (KRB)
135 mM NaCl
5 mM KCl
1 mM MgCl₂·6H₂O
0.4 mM KH₂PO₄
1 mM MgSO₄·7H₂O
20 mM HEPES
Adjust the pH to 7.4 with NaOH
Store at 4 °C
Add 1 mM CaCl₂ and 5.5 mM glucose, fresh before use
Digitonin lysis solution
100 μM digitonin
10 mM CaCl₂
Store at 4 °C for maximum 4 days

Acknowledgments

The research is supported by grants from the European Union (ERC mitoCalcium, No. 294777 to R.R.); Italian Ministries of Education, University and Research (PRIN to R.R., FIRB to R.R., FIRB Futuro in Ricerca RBFR10EGVP_002 to C.M.); NIH (grant 1P01AG025532-01A1 to R.R.) and French Muscular Dystrophy Association AFM-Téléthon (18857 to C.M.).

References

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Brini, M. (2008). Calcium-sensitive photoproteins. Methods 46(3): 160-166.
Brini, M., Marsault, R., Bastianutto, C., Alvarez, J., Pozzan, T. and Rizzuto, R. (1995). Transfected aequorin in the measurement of cytosolic Ca²⁺ concentration ([Ca²⁺]c). A critical evaluation. J Biol Chem 270(17): 9896-9903.
Granatiero, V., Patron, M., Tosatto, A., Merli, G. and Rizzuto, R. (2014a). Using targeted variants of aequorin to measure Ca²⁺ levels in intracellular organelles. Cold Spring Harb Protoc 2014(1): pdb-prot072843.
Granatiero, V., Patron, M., Tosatto, A., Merli, G. and Rizzuto, R. (2014b). The use of aequorin and its variants for Ca²⁺ measurements. Cold Spring Harb Protoc 2014(1): pdb-top066118.
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Head, J. F., Inouye, S., Teranishi, K. and Shimomura, O. (2000). The crystal structure of the photoprotein aequorin at 2.3 Å resolution. Nature 405(6784): 372-376.