- high-resolution terminology - matching measurements at high-resolution
Carboxy SNARF 1
Description
Carboxy SNARF® 1 is a cell-impermeant pH indicator dye. The pKa of ~7.5 makes it useful for measuring pH in the range of pH 7 to pH 8. The emission shifts from yellow-orange at low pH to deep red fluorescence at high pH. Ratiometric fluorometry, therefore, is applied at two emission wavelengths,such as 580 nm and 640 nm.
Relative molecular mass: Mr = 453.45
Abbreviation: SNARF
Reference: https://www.thermofisher.com/order/catalog/product/S22801
MitoPedia methods:
Fluorometry
- Life technologies (Invitrogen): "Stock solutions can be made in water or buffer and should be divided into aliquots and stored frozen at <=-20 °C, protected form light."
- Solution used by the producer for specific measurements: 50 mM potassium phosphate, pH 10.
- Solution used for measurement in patent of producer: 40 mM potassium phosphate, pH 9, probable just K2HPO4 solution.
- Since solubility in water at neutral pH is not known, a high pH>=9 solution should be used to ensure a proper solution state.
Carboxy SNARF 1 with the NextGen-O2k
Stock solution
- Lot Nr. 1189785 (2014-07-08)
- 1 mg in 1.1 mL 50 mM K2HPO4 solution (pH at RT ca 9.3) --> 2 mM
- 100 µL aliquots in 0.5 mL Eppendorf, store at -20 °C.
Calibration
- Calibration buffers
- >= 3 calibration points of known pH value are required.
- Basic endpoint: fluorophore completely deprotonated.
- Acidic endpoint: fluorophore completly protonated.
- The concept of calibration buffers was evaluated critically, because
- using different media for calibration ignores the changes in optical properties due to the sample;
- small differences in pH value are necessary that ideally should be measured in the chamber anyway.
- The concept of calibration buffers was evaluated critically, because
- Injection of acid / base during the experiment
- Inject small amounts of acid or base into the chamber and measure the resulting pH directly in the chamber with a potentiometric pH electrode. Repeated opening and closing of the chamber is necessary, since the pH electrode has to be removed during fluorometric measurement.
- For the calculations the fluorescence signal at the complete protonated and deprotonated forms of the fluorophore are required (endpoints). These states are established experimentally at the end of the experiment by injecting strong acids and bases. To avoid precipitation (and thereby changing optical properties) the following points have to be considered:
- Coagulation of cells in alkaline solution: see
- Precipitation of Mg(OH)2 and Ca(OH): Mg(OH)2 has the a lower solubility and is therefore the limiting factor of these two: KL Mg(OH)2 at ca 20° 10-11. c(Mg) in e.g. Hanks mod buffer ca 0.5 mM. Precipitations should start only above pH > 10. The limiting factor would thereby be the coagulation of cells (but also search KL for higher Temp).
- Precipitation of fluorophore at acidic conditions: the fluorophore precipitates when completely protonated. The acidic endpoint should therefore be measured last and immediately after injecting the acid.
