Fluorescein excitation shown as dotted line and emission shown as solid green histogram
5488530/30498517(in buffer) 1
(in antifade) 4		microscopy, flow cytometry, fluorescence microplate reader

Fluorescein (FITC) 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
  • A 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.

Protein Labeling Reagents Selection Guide (NHS ester, maleimide, etc.)  Search FITC secondary antibodies

Experimental data using Fluorescein (FITC)
multicolor imaging

Figure 1. HeLa cells were fixed and permeabilized using several Invitrogen products Image-iT Fixation/Permeabilization Kit then incubated with Anti-ATP Synthase Subunit IF1 and Goat anti-Mouse Highly Cross-Adsorbed Secondary Antibody, FITC. Cells were counterstained with NucBlue Live and ActinRed 555 then mounted using ProLong Gold Antifade Mountant and imaged using the EVOS M7000 Imaging System and a EVOS 40X Objective, coverslip-corrected

Figure 2. Flow cytometry analysis of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC was performed using HeLa cells stained with HDAC4 Recombinant Rabbit Polyclonal Antibody. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with HDAC4 Recombinant Rabbit Polyclonal Antibody or with rabbit isotype control at 3-5 µg/million cells in 2.5% BSA and incubated for 2 hours at room temperature. The cells were then labeled with Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC at a dilution of 1:500 for 1 hour at room temperature. A representative 10,000 cells were acquired and analyzed for each sample using the Attune NxT Acoustic Focusing Cytometer. Panels a, b and c represent cells stained with the secondary antibody alone, isotype control and HDAC4 Recombinant Rabbit Polyclonal Antibody respectively. Median fluorescence intensity from the three samples is compared in panel d.

Alexa Fluor 488 has greater fluorescence compared to FITC
Figure 3. Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates prepared from Alexa Fluor 488 dye and from fluorescein isothiocyanate (FITC). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of a reference dye and multiplying by the dye:protein labeling ratio.

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