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Calibration Protocol for Fluorescent Calcium Indicators

Calcium Calibration Buffers

Fura-2 Calcium Calibration
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.

Calcium Calibration Buffers *zero and 10 mM CaEGTA (2 x 50 mL)*
Zero Free Calcium Buffer50 mL10 mM EGTA in 100 mM KCl, 30 mM MOPS, pH 7.2
  • 2°C - 8°C
39 µM Free Calcium Buffer10 mM EGTA in 100 mM KCl, 30 mM MOPS, 10 mM CaCl2, pH 7.2
Calcium Indicator Calcium Imaging Calibration Buffers *zero to 10 mM CaEGTA, 50 µM calcium indicator (11 × 1 mL)*
Zero Free Calcium Buffer1 mL0 mM CaEGTA and 50 µM calcium indicator
  • 2°C - 8°C
  • Protect from light
0.017 µM Free Calcium Buffer1.0 mM CaEGTA and 50 µM calcium indicator
0.038 µM Free Calcium Buffer2.0 mM CaEGTA and 50 µM calcium indicator
0.065 µM Free Calcium Buffer3.0 mM CaEGTA and 50 µM calcium indicator
0.100 µM Free Calcium Buffer4.0 mM CaEGTA and 50 µM calcium indicator
0.150 µM Free Calcium Buffer5.0 mM CaEGTA and 50 µM calcium indicator
0.225 µM Free Calcium Buffer6.0 mM CaEGTA and 50 µM calcium indicator
0.351 µM Free Calcium Buffer7.0 mM CaEGTA and 50 µM calcium indicator
0.602 µM Free Calcium Buffer8.0 mM CaEGTA and 50 µM calcium indicator
1.35 µM Free Calcium Buffer9.0 mM CaEGTA and 50 µM calcium indicator
39 µM Free Calcium Buffer10.0 mM CaEGTA and 50 µM calcium indicator
Control Buffer, no calcium indicator10.0 mM CaEGTA (no calcium indicator)
  • 2°C - 8°C
  1. Prepared in 18 Mohm deionized water.
  2. In 10 mM K2EGTA, 100 mM KCl, 30 mM MOPS, pH 7.2, prepared in 18 Mohm deionized water. In addition, each vial contains 15 µm-diameter polystyrene beads in suspension at 16,000 beads per mL to serve as coverslip spacers and focusing aids.


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 IndicatorExcitation WavelengthEmission WavelengthProper Spectrum
    Fura-2Scan 300-450 nm490-520 nmExcitation
    Fura-8Scan 300-450 nm510-540 nmExcitation
    Indo-1340-360 nmscan >360 nmEmission
    Quin-2Scan >300 nm480-500 nmExcitation
    Fluo-3480-500 nmscan >500 nmEmission
    Fluo-4480-500 nmscan >500 nmEmission
    Fluo-8®480-500 nmscan >500 nmEmission
    Cal® 520480-500 nmscan >500 nmEmission
    Calbryte™ 520480-500 nmscan >500 nmEmission
    Cal™ 590560-580 nmscan >580 nmEmission
    Calbryte™ 590570-590 nmscan >590 nmEmission
    Cal™ 630600-620 nmscan >620 nmEmission
    Calbryte™ 630600-620 nmscan >620 nmEmission
    Rhod-2540-560 nmscan >560 nmEmission

  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+]freeVolume to replace using a 2 mL sample
    0 mM0 µM"zero Ca2+ sample"
    1 mM0.017 µMReplace 0.200 mL
    2 mM0.038 µMReplace 0.222 mL
    3 mM0.065 µMReplace 0.250 mL
    4 mM0.100 µMReplace 0.286 mL
    5 mM0.150 µMReplace 0.333 mL
    6 mM0.225 µMReplace 0.400 mL
    7 mM0.351 µMReplace 0.500 mL
    8 mM0.602 µMReplace 0.667 mL
    9 mM1.35 µMReplace 1.000 mL
    10 mM39 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 = KdEGTA 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:

[Ca2+]free=(150.5 x 10-9 M) x 0.11
 =1.67 x 10-8 M
 =0.0167 µM

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.

pH20 °C37 °C

Original created on April 27, 2023, last updated on April 27, 2023
Tagged under: Calcium Indicators, Calcium Calibration, Calibration Protocol