Fura salt can be purchased from Thermo Scientific (Fura-2 pentapotassium salt or Fura-4f pentapottasium salt).

Test solutions are typically made to mimic the intracellular condition, with high and low calcium solutions containing Fura and a background solution (either high or low calcium solution containing no Fura-2). The following recipes serve as examples: High Calcium (150mM KCl, 10mM NaCl, 3mM MgCl2, 10mM Hepes, 1μM Fura-2 pentapotassium salt, 1mM CaCl2, pH 7.4), Zero Calcium (150mM KCl, 10mM NaCl, 3mM MgCl2, 10mM Hepes, 1μM Fura-2 pentapotassium salt, 10mM EGTA pH 7.4).

Prepare the solutions by mixing all components except the Fura-2 in a volume that can be reliably pH'd (100mL). Separately, solubilize the Fura salt in water to a final concentration of 1mM. Add 1μL of the 1mM Fura-2 solution to 1mL of each test solution.

Place 5-8uL drops of the high and low calcium concentration with Fura salt and Fura-free background solution on a glass-bottom 35mm dish or imaging chamber. Alternatively, fluorescence recordings can be made from a volume of each solution in thin-walled capillary tubes.

Droplet method:

Step 1: Find drop with low calcium. Bring into focus on the edge of the drop, then move further inside the drop → Measure.

Step 2: Find drop with high calcium. Bring into focus on the edge of the drop, then move further inside the drop → Measure.

Step 3: Find drop with Fura-free solution for background measurement. Bring into focus on the edge of the drop, then move further inside the drop → Measure

Capillary method:

Step 1: Determine the background signal for the numerator and the denominator.

Step 2: Fill in these constants via Operations → Constants → Dual Excitation Numeric Background.

Step 3: Look at the Ratio in the low calcium solution (0.125)

Step 4: Look at the Ration in the high calcium solution (4.08)

Step 5: Calculate the dynamic range 4.08/0.125 = 32x. 

A good dynamic range is above 10x.