Calibration Protocol for Fluorescent Calcium Indicators

Calcium Calibration Buffers

Figure 1.Fluorescence excitation spectra of Fura-2 in solutions conctaining 0 to 39uM free Ca²⁺

Accurate measurement of calcium levels requires proper calibration of the Ca²⁺ indicator. To achieve this, calcium calibration buffers are utilized to determine the dissociation constant (Kd) of fluorescent Ca²⁺ indicators, considering factors such as temperature, pH, and ionic strength. The Kd of an ion indicator can be calculated using a laboratory fluorometer or quantitative imaging system by generating a plot from scanning the excitation or emission of the indicator at 11 different Ca²⁺ concentrations (as shown in Table 1).

To minimize errors in indicator concentration, the reciprocal dilution method is employed, which involves using 50 mL of 10 mM K₂EGTA and 50 mL of 10 mM CaEGTA, both containing 100 mM KCl and 30 mM MOPS at pH 7.2. These stock solutions can be blended to prepare buffers with free Ca²⁺ concentrations ranging from 0 μM to 39 μM, as depicted in Table 3.

Table 1. Buffers for proper calibration of calcium indicators.

Experimental Protocol for Calcium Indicator Calibration

Protocol for preparing reciprocal dilutions

1. Create a stock solution of the Ca²⁺ indicator (in salt form) using a dilute buffer free of both Ca²⁺ and EGTA. The concentration of the stock solution should be between 100-500 times higher than the actual measurement concentration required, which is usually in the range of 0.2-1 millimoles per liter.
2. Create the "zero Ca²⁺ sample" by adding a small portion of the stock indicator solution to 2 mL of Zero Free Calcium Buffer. This will result in an indicator concentration of approximately 1-10 μM. Note that while any sample volume can be used, this example uses a 2 mL sample volume.
3. To obtain a complete series of dilutions, it is necessary to use a larger volume of the high Ca²⁺ buffer. To prepare the "high Ca²⁺ sample," you should dilute precisely three times the amount of dye into 6 mL of 39 μM Free Calcium Buffer.
4. Check that the pH of both solutions is the same and take note of the pH value to the nearest 0.01 units.
5. To take measurements using a fluorometer, carefully pour precisely 2 mL of the "zero Ca²⁺ sample" into a cuvette and then record the corresponding spectrum. The appropriate wavelengths may vary depending on the instrument, but Table 2 provides a reference for the recommended wavelengths.
   

   Table 2. Parameters for measuring the spectrum of fluorescent Ca²⁺ indicators.

   Calcium Indicator	Excitation Wavelength	Emission Wavelength	Proper Spectrum
   Fura-2	Scan 300-450 nm	490-520 nm	Excitation
   Fura-8	Scan 300-450 nm	510-540 nm	Excitation
   Indo-1	340-360 nm	scan >360 nm	Emission
   Quin-2	Scan >300 nm	480-500 nm	Excitation
   Fluo-3	480-500 nm	scan >500 nm	Emission
   Fluo-4	480-500 nm	scan >500 nm	Emission
   Fluo-8®	480-500 nm	scan >500 nm	Emission
   Cal® 520	480-500 nm	scan >500 nm	Emission
   Calbryte™ 520	480-500 nm	scan >500 nm	Emission
   Cal™ 590	560-580 nm	scan >580 nm	Emission
   Calbryte™ 590	570-590 nm	scan >590 nm	Emission
   Cal™ 630	600-620 nm	scan >620 nm	Emission
   Calbryte™ 630	600-620 nm	scan >620 nm	Emission
   Rhod-2	540-560 nm	scan >560 nm	Emission

   
   
   
6. To prepare the next solution, take the initial sample with 0 mM CaEGTA/indicator in the cuvette and remove 0.2 mL from it. Next, add an equal amount of the "high Ca²⁺ sample" (0.2 mL) to the sample. This will increase the CaEGTA concentration to 1 mM, and the [Ca²⁺]_free will reach approximately 0.017 μM. The concentration of the dye or total EGTA will remain unchanged. You can use the following equation to determine the volume to remove and replace:

   Volume to remove/replace = (sample volume) × {(b – a)/(c – a)}

   • a = current mM CaEGTA
   • b = desired mM CaEGTA
   • c = mM CaEGTA in "high Ca²⁺ sample" (typically 10.0 mM CaEGTA)

   For this first dilution from 0 mM to 1 mM CaEGTA in a 2 mL sample, the replacement volume is calculated using the equation above as follows:

   2 mL x (1 mM - 0 mM)/(10 mM -0 mM) = 0.2 mL

7. Scan the spectrum again, then remove another aliquot (this time 0.22 mL) and replace it with 0.22 mL of the "high Ca²⁺ sample." This will result in a solution with 2 mM CaEGTA and a [Ca²⁺]_free of approximately 0.038 μM.
8. Record the spectrum. Then, prepare the indicator solutions with CaEGTA concentrations of 3, 4, 5, 6, 7, 8, and 9 mM using the same procedure as for the previous spectrum (Table 3), and always start with the previous solution. For the 10 mM CaEGTA spectrum, replace the previous measurement sample with 2 mL from the "high Ca²⁺ sample." Avoid over-illuminating the solutions when obtaining spectra. The quality of the dilutions and measurements will be evident for indicators such as fura-2 or indo-1 that undergo excitation or emission shifts upon Ca²⁺ binding. They will exhibit a clean isosbestic point if the dilutions are accurate.

   Table 3. Parameters for measuring the spectrum of fluorescent Ca²⁺ indicators.

   CaEGTA	[Ca2+]free	Volume to replace using a 2 mL sample
   0 mM	0 µM	"zero Ca2+ sample"
   1 mM	0.017 µM	Replace 0.200 mL
   2 mM	0.038 µM	Replace 0.222 mL
   3 mM	0.065 µM	Replace 0.250 mL
   4 mM	0.100 µM	Replace 0.286 mL
   5 mM	0.150 µM	Replace 0.333 mL
   6 mM	0.225 µM	Replace 0.400 mL
   7 mM	0.351 µM	Replace 0.500 mL
   8 mM	0.602 µM	Replace 0.667 mL
   9 mM	1.35 µM	Replace 1.000 mL
   10 mM	39 µM	"high Ca2+ sample"

Calculating free Ca²⁺ concentrations

To determine the [Ca²⁺]_free value for each solution, it is necessary to perform a calculation since this value is very small in the calibration buffers. To do this, multiply the Kd of EGTA for Ca²⁺ (at the appropriate pH, ionic strength, and temperature) by the molar ratio of CaEGTA to K₂EGTA in the particular solution. For instance, in the first dilution, the [Ca²⁺]_free is increased from almost zero to about 0.017 μM by replacing 200 μL of 10 mM K₂EGTA with an equal volume of 10 mM CaEGTA. The [Ca²⁺]_free value can be calculated using the following equation based on the Kd of EGTA for Ca²⁺:

[Ca²⁺]_free = K_d^EGTA x [CaEGTA]/[K₂EGTA]

The ratio of CaEGTA to K₂EGTA in the 1 mM CaEGTA solution is 1:9 or 0.11. This value is multiplied by the Kd EGTA at the pH, ionic strength, and temperature at which the measurement is made (Table 4). Using the reagents outlined in Table 1 (pH 7.2 with an ionic strength of 100 mM KCl) at 20°C, the Kd EGTA is 150.5 × 10−9 M. Therefore, for the 1 mM CaEGTA solution (with 9 mM K₂EGTA also present), the [Ca²⁺]_free is:

Table 3 shows the values of [Ca²⁺]_free at 20°C for solutions with a pH of 7.20 and ionic strength of 100 mM KCl. It's important to note that the affinity of EGTA, which is used to buffer Ca²⁺ in these solutions, is highly dependent on pH, ionic strength, and temperature, as indicated in Table 4. Even a slight change of 0.05 pH units can cause up to a 20% alteration in the Kd EGTA value. Therefore, if your measurement is conducted under conditions that significantly differ from those in Table 4, it's crucial to make corrections to obtain the accurate Kd EGTA value for Ca²⁺. A comprehensive review by Bers and colleagues in Volume 40 of the Methods in Cell Biology series explains the methods for performing these corrections. Additionally, the study by Groden and colleagues demonstrates the impact of Kd EGTA corrections on Ca²⁺ measurements using fura-2.

Plotting the data

Once you've recorded the spectra, you can plot the excitation or emission at a single wavelength against [Ca²⁺]_free to generate a calibration curve. This curve can then be used to determine the [Ca²⁺]_free of an unknown solution. For ratioable indicators like fura-2 or indo-1, you can plot the ratio of the absorption, excitation, or emission at two wavelengths against [Ca²⁺]_free. By doing so, you can minimize the effects of artifacts caused by variations in indicator concentration, photobleaching, and path length, since these factors tend to have a similar effect on intensities at both wavelengths and cancel out in the ratio of intensities. However, using ratio techniques makes calculations of Kd Indicator slightly more complex, as described in detail in reference 4.

Raw spectral data and the accompanying data analysis obtained with the calcium calibration buffers in Table 1 and fura-2 are shown in Figure 1. The data is plotted as the log of the [Ca²⁺]_free (x-axis) versus the log {(F − Fmin)/(Fmax − F)} (y-axis). This double log plot gives an x-intercept that is the log of the Kd Indicator expressed in moles/liter. In the example, the x-intercept is −6.84. The inverse log of this number is 145 × 10−9 M (145 nM). The slope of the plot is 1.0, which reflects the 1:1 binding of each fura-2 with a single Ca²⁺ ion. The first dilution (from "zero" Ca²⁺ to 0.017 μM) has the greatest error due to contaminating ions from glassware, reagents, etc., and may sometimes be unreliable.

Table 4. Dissociation constants of EGTA for Ca²⁺ in 0.1 M KCl.