CAS Number: N/A Alternative Name: FARD
FITC-Antonia Red-dextran (FARD) is a dextran derivative that is labelled with two fluorophores, FITC and Antonia Red (see Fig 1) and it is currently produced at a molecular weight of 20 kDa.
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Structure and physical properties
Structurally, each of the fluorophores (FITC and Antonia Red) are bound to dextran through thiourethane linkages (Fig 1). The degree of substitution (DS) lies in the range of 0.001 to 0.020 mol of dye per mole of glucose units for each of the fluorophores. The molar ratio of FITC/AR bound to dextran can vary between 1:1 and 1:3 and it is assessed spectrophotometrically at a neutral pH.
As a result of the dual fluorescent labelling of dextran, FITC-Antonia Red-dextran exhibits fluorescence emission at λfl=517±5nm when photoexcited at λex=493 nm (due to FITC) and at λfl=600±5nm when photoexcited at λex=585±5nm nm (due to AR).
What renders this TdB Labs product unique is that the fluorescence (as well as the absorbance) of one of the fluorophores (FITC) is highly dependent on pH while the corresponding ones of the other fluorophore (AR) remains nearly unchanged between pH 2 and 10 (see Fig 2). This allows for accurate determination of pH in living cells or tissues and thereby renders this product an invaluable tool for intravital microscopy.
In the series of experiments involving FARD20 (pH-probe) in aqueous solutions, fluorescence spectra of both the fluorophores were recorded simultaneously (FITC: green fluorescence centered at 520 nm and Antonia RedTM: red fluorescence centered at 600 nm) while varying pH within the values 3.0 and 8.0 (Fig 3). Labbot helped us visualize the pH-dependent nature of FITC (“working fluorophore”) and the pH-independent nature of Antonia RedTM.
Plotting the ratio of fluorescence intensity of FITC over that of Antonia RedTM against pH results in a characteristic sigmoidal curve (Fig 4) which describes the performance and pH-responsiveness of the FARD20 pH-probe. A pKa value of 5.91 was determined, corresponding to the standard behaviour of FITC (working fluorophore). A good fitting of the recorded data to Henderson-Hasselbalch equation denotes that the pH of a FARD20 solution is equal to pKa=5.91 when concentrations of the acid and its conjugate base FITC forms are equal (see Fig 5). Noteworthy, pKa of the acidic and basic forms of fluorescein bound on FARD20 (monoanionic and dianionic respectively) is somewhat lower compared to “free” fluorescein (pKa=6.5) denoting a shift of the sigmoidal curve towards lower pH and thus increasing span of the ratiometric activity of FARD20 by approx. 0.6 pH units.
Storage and stability
FARD20 should be stored in a well-sealed container in the dark, at room temperature.
FARD20 is delivered as a light brown to dark purple powder or crystals readily soluble in water and DMSO. However, it is incompatible with strong oxidizing agents.
FARD20 is suitable for applications requiring accurate detection and monitoring of pH in living cells or tissues, where it can operate at a wide range of pH spanning from 3.5 to 8.0.
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