Fluorescein Isothiocyanate

Fluorescein isothiocyanate (FITC) can be given through intratracheal administration to promote a fibrotic response.29 While this chemical is not associated with human disease, it produces a prolonged fibrotic response, up to 6 months, which makes it potentially useful in studying the mechanisms of the fibrotic response.7 Following administration, this agent leads to an early inflammatory response, followed by the development of fibrosis that is patchy. Because FITC is fluorescent, areas of deposition and injury can be viewed via microscopy.7,30 A limitation of the model is that the variability in dosing limits its reproducibility.

 

 

Fluorescein Isothiocyanate (FITC)

The FITC model of lung fibrosis was discovered fortuitously when investigators were injecting FITC into the lung in hopes of labeling dendritic cells (DCs) which would then allow for tracking of the DC migration to the lymph nodes; however, investigators noted that these mice also developed lung fibrosis (Roberts et al., 1995). The i.t. delivery of FITC results in alveolar and vascular permeability resulting in lung fibrosis within 14–21 days (Moore and Hogaboam, 2008; Moore et al., 2001). This model is particularly useful when investigators want to visualize the exact location of the injury as FITC conjugates to tissue within the lung where it initially deposits. This allows use of immunofluorescence to localize areas of initial injury and previous studies have shown that the initial areas of injury correlate well with areas of eventual fibrosis deposition (Moore and Hogaboam, 2008). Doses ranging from 0.007 mg g− 1 body weight dissolved in saline to i.t. delivery of 50 μL of a 1.4 mg mL− 1 solution have been used in mice (Christensen et al., 1999; Moore et al., 2005; Roberts et al., 1995). It is imperative that the solution is made fresh for each instillation and the degree of sonication matters, as particles that are too large do not cause fibrosis, but particles that are too fine can cause excessive lung injury and death (Moore and Hogaboam, 2008). Similar to what was noted before with bleomycin

 

, the source of FITC can vary in the degree of fibrosis that is generated; thus dose-finding studies are necessary, and we recommend that sonication not be more than 10 s on ice. Both Balb/c and C57Bl/6 mice develop a persistent fibrotic response, dependent on Th2 cytokines, lasting for months (Moore and Hogaboam, 2008; Kolodsick et al., 2004). Our laboratory has also shown that lung fibrosis induced by FITC is regulated by CCL12-mediated recruitment of fibrocytes (Moore et al., 2006). To our knowledge, the bleomycin and FITC models of lung fibrosis appear to be regulated by similar inflammatory and pro-fibrotic mediators. One potential advantage for the FITC model is that the deposition of FITC, and thus, the persistence of the fibrotic response is more long-lasting than with bleomycin. For instance, we have noted FITC-mediated fibrosis to persist for more than 4 months.

FITC used for Excitation and Emission in Flow Cytometry
FITC used for Excitation and Emission in Flow Cytometry

 

In flow cytometry, laser light is usually used to excite the fluorochromes. These lasers produce light in the UV and/or visible range. Fluorochromes are selected based on their abilities to fluoresce with the wavelengths of light produced by the lasers. Therefore, if a flow cytometer has only one laser that produces only 488 nm light, then only fluorochromes that are excited by 488 nm light can be used. The chemical properties of the fluorochrome determine whether its electrons can be excited to the higher energy state by a specific wavelength of laser light. If the electrons can be excited to the higher energy state, the chemical properties of the fluorochrome will also determine the amount of energy lost as heat when the electrons drop back down to the lowest singlet excited state and the wavelength of light produced when the electrons return to their ground state.

The electrons of a fluorochrome can be excited by a range of wavelengths of light. For example, the fluorochrome, fluorescein, will fluoresce when hit by light with a wavelength between 430 nm and 520 nm. However, the closer the excitation wavelength is to 495 nm, the more fluorescence will be produced. This optimal wavelength is called the excitation peak. Similarly, the light produced by fluorochromes has a range of wavelengths. The emission of light from fluorescein, ranges from 490 nm to 630 nm, and the emission peak is approximately 520 nm.

Fluorochrome Selection

Knowing the excitation and emission properties of fluorescent compounds makes it possible to select combinations of fluorochromes that will work together optimally on a specific flow cytometer with specific lasers. However, for a fluorochrome to be useful in a biological application, it must attach to or be contained within a particle of biological significance. Some fluorochromes are useful because they bind to specific chemical structures, such as antibodies or the nucleic acids in DNA or RNA.

Fluorochromes that are used most often in flow cytometry are ones that attach in some way to biologically significant molecules and are excitable by the lasers that are commonly found on commercial flow cytometers. Many fluorochromes can be attached to antibodies, which will then bind to specific chemical structures on or inside of cells. If these chemical structures are unique to a specific type of cell, then the fluorochrome will identify that cell type. This technique of identifying cells using fluorescent antibodies is called immunophenotyping.

A list of the fluorochromes used most often in immunophenotyping is shown in Table 1 with their peak excitation and emission wavelengths and the laser wavelengths most often used to excite them on a flow cytometer. Table 2 shows the lasers that can generate the required wavelengths of light to excite the various fluorochromes. Some other common applications of fluorochromes in flow cytometry include the detection of intracellular calcium, measurement of the relative amount of cellular DNA or RNA, and measurement of transcription levels using a fluorescent protein as a reporter gene.

Fluorescein derivatives are the most common fluorescent reagents for biological research because of their high absorptivity, excellent fluorescence quantum yield, and good water solubility.

Fluorescein-based dyes and their conjugates have several performance characteristics that may facilitate or limit use in certain applications. Fluorescein displays:

  • A relatively high rate of photobleaching
  • Fluorescent signal that is sensitive to pH changes
  • A relatively broad fluorescence emission spectrum
  • Fluorescence quenching on conjugation to biopolymers
We offer a broad range of fluorescein conjugates and derivatives as well as a series of high-performance fluorophores with improved characteristics for labeling and detection.

 

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