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Fluorescein isothiocyanate (FITC)

What is FITC used for?


FITC (fluorescein isothiocyanate) is widely used to label proteins, antibodies, peptides, hormones, amine-modified oligonucleotides, and other amine-containing molecules with the green fluorescent dye fluorescein. The isothiocyanate moiety (-N=C=S) on FITC reacts with amino-terminal and primary amine groups on the target biomolecule to form a covalent thiourea bond. The resulting fluorescein conjugates can be used as specific probes in several applications, including enzymatic kinetics, immunocytochemistry, immunohistochemistry, fluorescence microscopy, flow cytometry, and fluorescence in situ hybridization. FITC has an excitation maximum of 491 nm which closely matches the 488 nm spectral line found in most instruments, and a relatively broad emission spectrum with a maximum of 516 nm. (11)


Tubulin Stain
Figure 1. HeLa cells were incubated with rabbit anti-tubulin followed by FITC goat anti-rabbit IgG (H+L) (Cat No. 16868). Cell nuclei were counter-stained using Hoechst 33342 (Cat No. 17530).

 

Is FITC the same as fluorescein?


Although FITC (Ex/Em= 491/516 nm) and fluorescein (Ex/Em= 498/517 nm) exhibit similar spectral profiles, they are not equivalent. FITC is a fluorescein derivative consisting of a fluorescein core and an amine-reactive isothiocyanate moiety. The isothiocyanate group reacts with primary amines forming covalent thiourea bonds that attach fluorescein to the biomolecule. Moreover, FITC is typically supplied as a mix of isomers consisting of fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). (10)



Chemical structure of fluorescein and FITC
Figure 2. Chemical structures of fluorescein (left) and FITC (right). Outlined in red is the reactive isothiocyanate moiety.

 

How does FITC bind to proteins?


FITC reacts with free primary amine groups of proteins to form covalent bonds. The isothiocyanate (-N=C=S) group is reactive towards any nucleophilic site, but FITC will selectively react with N-terminal amines because these bonds are far more stable. The primary amine attacks the electrophilic carbon of the isothiocyanate group, forming a thiourea bond connecting FITC and the protein in question. Conditions such as pH must be controlled, as the isothiocyanate group technically can react with secondary amines and other nucleophiles depending on reaction conditions. (9)


Figure 3. FITC labeling reaction. FITC conjugation occurs through the free amino groups of proteins or peptides, forming a stable thiourea bond.

 

How do I determine the number of FITC dyes labeled to a protein?


The degree of labeling (DOL) is a method commonly used to determine the average number of fluorophores, such as FITC, that are labeled to a protein. It is calculated by determining the conjugate's protein and fluorophore molar concentrations based on absorbance measurements. These concentrations are then expressed as a molar ratio in the form of label:protein. For antibodies, the ideal DOL usually falls between 2 to 10. However, a more precise value will largely depend on the properties of the label and protein. This means that for many bioconjugations, the optimal DOL must be experimentally determined, often through several small-batch labelings. Before the DOL can be determined, the following specifications must be known (7):

  • The molar extinction coefficient (ε) of the unlabeled protein (for IgG ε = 210,000 M-1cm-1)
  • The molar extinction coefficient and absorbance maximum (Amax) of the fluorophore (for FITC ε = 73,000 M-1cm-1, Amax = 516 nm)
  • The correction factor at 280 nm (A280) of the fluorophore (for FITC A280 = 0.254)

The following protocol can be used to determine the degree of labeling:

  1. Remove all excess, unbound dye by dialysis or gel filtration.
  2. Measure the absorbance of the dye:protein conjugate at 280 nm in a spectrophotometer cuvette with a 1 cm path length.
  3. Measure the absorbance of the dye:protein conjugate at the absorbance maximum of the fluorophore.
  4. Calculate the molarity of the protein using the following formula:

    Protein Concentration (M) = ((A280 - (Amax x CF) ÷ ε) x Dilution factor

    • ε = protein molar extinction coefficient
    • Amax = absorbance of dye measure at wavelength maximum
    • CF = correction factor
    • Dilution factor = Amount sample was diluted before absorbance measurement
  5. Calculate the degree of labeling using the following formula:

    Moles dye per mole protein = (Amax of labeled protein ÷ (ε* x protein concentration (M))) x Dilution factor

    • ε* = dye molar extinction coefficient

 

Is iFluor™ 488 and Alexa Fluor® 488 the same as FITC?


iFluor™ 488, Alexa Fluor® 488, and FITC are green fluorescent dyes with nearly identical spectral profiles, extinction coefficients, and fluorescence quantum yields (see table below). Like FITC, iFluor 488™ succinimidyl ester and Alexa Fluor® 488 NHS ester label the primary amines of proteins, antibodies, and other amine-containing molecules with their respective dyes. Because of these spectral similarities, either iFluor™ 488 or Alexa Fluor® 488 can seamlessly replace FITC in any application without having to adjust the experimental setup.

iFluor™ 488 and Alexa Fluor® 488 outshine FITC in all performance areas. Unlike FITC, which is susceptible to self-quenching, iFluor™ 488 and Alexa Fluor® 488 can be labeled to proteins at high molar ratios with minimal self-quenching resulting in brighter, more sensitive conjugates. iFluor™ 488 and Alexa Fluor® 488 labeled probes are highly photostable and insensitive to changes in pH, whereas FITC exhibits significant decreases in fluorescence intensity as the pH decreases from 10 to 3. iFluor™ 488 and Alexa Fluor® 488 are trademarked products of AAT Bioquest and ThermoFisher, respectively. (4)


iFluor488 and Alexa Fluor 488 comparison
Figure 4. HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by iFluor™ 488 goat anti-mouse IgG conjugate (Green, Left) or Alexa Fluor® 488 goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Cat No. 17530).

 

Table 1. Spectral characteristics of iFluor™ 488, Alexa Fluor® 488 and FITC

Dye Ex (nm) Em (nm) Spectrum Extinction Coefficient (M-1cm-1) Quantum Yield Unit Size Cat No.
iFluor™ 488 491 516 75,000 0.9 1 mg 1023
XFD488 *Alexa Fluor® 488 equivalent* 499 520 73,000 0.92 1 mg 1812
FITC 491 516 73,000 0.92 1 g 121

 

What is a FITC filter?


Filters are used in fluorescence imaging and microscopy to improve image quality by enhancing or blocking specific wavelengths. These filters collect light more efficiently by blocking wavelengths that are not useful in the experiment. The excitation source light may be filtered before hitting the sample to reduce noise or focus on specific incoming wavelengths in cases where more than one fluorescent compound is being used. The emitted light is also filtered so that the detector only analyzes the regions of concern, making experimentation more efficient. A commercial FITC filter set typically consists of three optical filters -an excitation filter, emission filter, and dichroic beamsplitter - optimized for detecting FITC fluorescence and other green fluorescent dyes, including iFluor™ 488. The excitation and emission filters are bandpass filters that transmit a wavelength range that corresponds only to the excitation and emission spectra of FITC. For example, a FITC filter set might have an excitation and emission filter with a wavelength range of 467-498 nm and 513-556 nm, respectively. (6)

 

Figure 5. Example of filter set optimized to detect FITC fluorescence.

 

Is FITC cell-permeable?


No, FITC is not cell-permeable. This isothiocyanate derivative of fluorescein cannot penetrate healthy membranes of live cells. Instead, when incubated with live cells, FITC will bind to any free amine groups on membrane proteins exposed to the extracellular matrix, resulting in live cells with dimly fluorescent membranes. (8)

 

Is FITC sensitive to temperature and pH?


Yes, FITC is sensitive to both temperature and pH. It has been well-documented that the fluorescent signal intensity of FITC-labeled conjugates decreases as the environment becomes more acidic. For example, the intensity of FITC-dextran decreases by more than 95% as pH is reduced from 10 to 3. Temperature, on the other hand, affects the stability of FITC. At elevated temperatures, FITC is very unstable, and conjugates produced with FITC are susceptible to hydrolysis of the fluorescein label. (5)

 

How do you make a FITC solution?


FITC is generally supplied as a lyophilized solid and must be resuspended in an organic solvent, such as ethanol, DMSO, or DMF, before labeling proteins or any other amine-containing biomolecule. To make a FITC stock solution, reconstitute the dye in high-quality, anhydrous DMSO, such that the concentration is approximately 1 mg/mL (i.e., for optimal results, this should be prepared fresh for each labeling reaction). Further dilution of the FITC stock solution into an aqueous buffer (e.g., HBSS) is required before conjugation. We do not recommend storing FITC in aqueous solutions as it is unstable in water. (2)

 

Table 2. FITC stock solution preparation. The volume of DMSO needed to reconstitute a specific mass of FITC to a given concentration. Note that volume is only for preparing stock solutions. Refer to manufacture protocol for appropriate experimental buffers.

  0.1 mg 0.5 mg 1 mg 5 mg 10 mg
1 mM 256.819 µL 1.284 mL 2.568 mL 12.841 mL 25.682 mL
5 mM 51.364 µL 256.819 µL 513.637 µL 2.568 mL 5.136 mL
10 mM 25.682 µL 128.409 µL 256.819 µL 1.284 mL 2.568 mL

 

How do I store FITC?


FITC, as a lyophilized solid, should be stored refrigerated (2 °C to 8 °C), desiccated, and protected from light. Under these conditions, FITC should be stable for up to 12 months. In solvents such as DMSO, FITC can be stored at -80 °C for six months or -20 °C for one month, protected from light and under nitrogen. Never store FITC in aqueous media as it is unstable in water. (1)

Link: FITC datasheet

 

Can I use FITC together with GFP (green fluorescent protein)?


No, avoid using FITC and GFP together in the same application. FITC and GFP have nearly identical spectral profiles, making it virtually impossible to distinguish their fluorescence signals from one another due to significant cross-talk. It is best to use fluorophores with minimal spectral overlap for multicolor experiments. For example, green fluorescent dyes, such as FITC, pair well with red fluorescent dyes, such as iFluor™ 647 or allophycocyanin (APC). (3)

 

Figure 6. Left: Spectra comparison of FITC and GFP illustrating low compatibility due to significant cross-talk between them. Right: Spectra comparison of FITC and APC shows minimal overlap in their emission spectra, indicating FITC and APC are compatible for multicolor applications.

 

Can I use FITC with Phycoerythrin (PE) and Allophycocyanin (APC)?


Yes, FITC can be used with phycoerythrin (PE) and allophycocyanin (APC) for multicolor analysis. The caveat, however, is compensation using single-stained controls will be required during data analysis since two or more fluorophores have overlapping emission spectra. Compensation calculates how much interference a fluorophore will have in a channel not assigned specifically to measure it and prevents data misinterpretations by removing such artifacts. For the multicolor panel of FITC, PE, and APC, following excitation with the blue laser (488 nm), FITC emits a broad emission that spills into the PE detector. Additionally, PE shows spillover into the APC detector. The portion of the FITC spectrum detected in the PE detector and the PE spectrum detected in the APC detector must be subtracted from the PE and APC signals using compensation. (3)

 

Figure 7. Absorption and emission spectra of FITC (Green), PE (Yellow), and APC (Red).

 

References


  1. Braun, R. K., Rudnicki, M. A., Sekaly, R. P., & Filion, L. G. (2007). Filter selection for five-color flow cytometric analysis with a single laser. International journal of laboratory hematology, 29(5), 369-376. https://doi.org/10.1111/j.1365-2257.2006.00852.x
  2. Chaganti, L. K., Venkatakrishnan, N., & Bose, K. (2018). An efficient method for FITC labelling of proteins using tandem affinity purification. Bioscience reports, 38(6), BSR20181764. https://doi.org/10.1042/BSR20181764
  3. Chevrier, S., Crowell, H. L., Zanotelli, V., Engler, S., Robinson, M. D., & Bodenmiller, B. (2018). Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry. Cell systems, 6(5), 612-620.e5. https://doi.org/10.1016/j.cels.2018.02.010
  4. Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., & Zhuang, X. (2011). Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature methods, 8(12), 1027-1036. https://doi.org/10.1038/nmeth.1768
  5. Ma, L. Y., Wang, H. Y., Xie, H., & Xu, L. X. (2004). A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 60(8-9), 1865-1872. https://doi.org/10.1016/j.saa.2003.10.004
  6. McKinnon K. M. (2018). Multiparameter Conventional Flow Cytometry. Methods in molecular biology (Clifton, N.J.), 1678, 139-150. https://doi.org/10.1007/978-1-4939-7346-0_8
  7. McKinney, R., Thacker, L., & Hebert, G. A. (1976). Conjugation methods in immunofluorescence. Journal of dental research, 55, A38-A44. https://doi.org/10.1177/002203457605500117011
  8. Perfetto, S. P., Chattopadhyay, P. K., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R. A., & Roederer, M. (2010). Amine-reactive dyes for dead cell discrimination in fixed samples. Current protocols in cytometry, Chapter 9, Unit-9.34. https://doi.org/10.1002/0471142956.cy0934s53
  9. Shah, D., Guo, Y., Ocando, J., & Shao, J. (2019). FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer. Journal of pharmaceutical analysis, 9(6), 400-405. https://doi.org/10.1016/j.jpha.2019.08.002
  10. Takai, H., Kato, A., Nakamura, T., Tachibana, T., Sakurai, T., Nanami, M., & Suzuki, M. (2011). The importance of characterization of FITC-labeled antibodies used in tissue cross-reactivity studies. Acta histochemica, 113(4), 472-476. https://doi.org/10.1016/j.acthis.2010.04.007
  11. The, T. H., & Feltkamp, T. E. (1970). Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology, 18(6), 865-873.


Original created on June 28, 2022, last updated on June 28, 2022
Tagged under: FITC, FITC Spectra, Protein labeling, Fluorescein isothiocyanate, Fluorescein isothiocyanate color, Fluorescein isothiocyanate wavelength