Testing if the hardware involved for fluorescence measurements is working correctly is difficult if you need to use living cells loaded with Fura. Testing the dynamic range of the Fura signal and therefore all components of the fluorescence hardware without cells is a reliable measurement to validate the system.
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
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.
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
Step 1: Focus on lumen of capillary with low calcium solution → Measure.
Step 2: Focus on lumen of capillary with high calcium solution → Measure.
Step 3: Focus on lumen of capillary with Fura-free solution for background measurement → Measure
The raw data should look similar to this (although the PMT values should not exceed the linear range of the device, typically ~6000 counts).
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.
The dynamic range is far above the desired 10x. This indicates that the hardware for fluorescence measurements is working as it should. If you do not get decent calcium traces with cells the loading protocol needs adaptation.