Quantitative comparison of iFluor™ 488- and FITC- conjugated antibodies for use in cell labeling

The use of fluorophore labeled antibodies has been a powerful tool for the study of biological systems. These fluorophore-antibody conjugates have found important applications in ELISA, Western blotting, FACS and fluorescence microscopy. Their importance is attributed to the sensitivity with which they detect a substrate and the ease by which that detection event can be translated into a quantifiable signal (ie. fluorescence). Because of their importance in biological assays, a great deal of research has been conducted to identify and synthesize the ideal fluorophore for a given application. While subject to some debate, ideal fluorophores typically possess one or more of the following qualities: (1) strong fluorescence as characterized by a high extinction coefficient and quantum yield (2) high photostability and resistance to photobleaching (3) resilience under different environmental conditions, such as pH (4) minimal background interference or auto-fluorescence and (5) simplicity and consistency in experimental usage.

Here, we describe a novel fluorophore for cell labeling applications, iFluor^® 488. This fluorophore is part of the iFluor^® family that spans the spectrum from ultra-violet to infrared. iFluor^® 488 absorbs light at 491 nm and emits at 518 nm, making it spectrally similar to the conventional fluorophore fluorescein isothiocyanate (FITC), which absorbs light at 495 nm and emits at 521 nm. However, iFluor^® 488 is far superior to FITC for cell labeling applications, particularly when comparing fluorescence intensity and photostability. In the present study, iFluor^® 488 and FITC were conjugated to goat anti-mouse polyclonal antibodies (GAM). The resulting conjugates were used to perform cell stains for fluorescence microscopy. Comparisons were made for fluorescence intensity, degree of labeling (ie. the number of fluorophores per antibody) and photostability.

Materials and Methods

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Conjugation of FITC and iFluor^® 488 to GAM IgG

FITC and iFluor^® 488 (AAT Bioquest, Sunnyvale, CA) were conjugated to goat anti-mouse IgG (AAT Bioquest, Sunnyvale, CA) according to manufacturer's specifications. Final bioconjugates were purified using size exclusion spin columns (Bio-rad, Hercules, CA).

Determination of degree of labeling (DOL)

Degree of labeling was determined using absorbance measurements and Degree of Labeling Calculator (AAT Bioquest, Sunnyvale, CA).

Preparation of fixed cells

HeLa cells were seeded in a 96-well black wall/clear bottom plate (10,000 cells/well) in culture medium overnight. Growth medium was removed, cells were washed once with phosphate buffered saline (PBS) and then PBS was aspirated. 100 µL 4% paraformaldehyde in PBS (pH 7.4) was added to each well and incubated with cells for 15 minutes at room temperature to fix the cells. Cells were then washed with 200 µL PBS/well for three times. After fixation, 100 µL of blocking solution (PBS + 1% BSA) was added to each well to block for 30 minutes at room temperature. Blocking solution was then replaced with cell permeabilization buffer (PBS + 1% BSA + 0.1% TritonX-100 cells) and cells were incubated for 15 minutes at room temperature. Cells were then washed with 200 µL PBS/well for three times. 200 µL PBS was added to each well and the plate was stored at 4°C until staining.

Tubulin monoclonal antibody staining

Storage PBS buffer in each well was aspirated and 1 µg/mL alpha tubulin monoclonal antibody (Thermo Fisher, Waltham, MA) in PBS + 0.02% Tween-20 was added and incubated at room temperature for 30 minutes. For experimental control, cells were stained with 1 µg/mL mouse IgG. After incubation, cells were washed with 200 µL PBS/well for three times.

GAM IgG-FITC and GAM IgG-iFluor^® 488 staining

10 µg/mL of previously prepared GAM IgG-FITC was added to wells stained with alpha tubulin monoclonal antibody or stained with mouse IgG (control) for 30 minutes at room temperature. Cells were then washed with 200 µL PBS/well for three times. After washing, 100µL PBS was added to each well. The same procedure was repeated for GAM IgG-iFluor^® 488. Cells were imaged using a Keyence X710 fluorescence microscope with FITC channel.

Test of photobleaching and photostability

Cells were continuously illuminated using a Keyence X710 fluorescence microscope. Images of the cells were captured and saved every 2.5 seconds for the duration of 300 seconds. Final intensity values were determined using ImageJ software's integrated density (National Institutes of Health, Bethesda, MD).

Results

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Degree of labeling (DOL)

The degree of labeling (DOL) was calculated using AAT Bioquest's Degree of Labeling Calculator. The DOL represents the average moles of fluorophore per mole of antibody. For GAM IgG-FITC, the DOL was determined to be 4.5 fluorophores/antibody. For GAM IgG-iFluor^® 488, the DOL was determined to be 6.5 fluorophores/antibody.

Photostability

Measurements of total fluorescence intensity (TFI) showed that HeLa cells labeled with GAM IgG-FITC had an initial intensity of 449.33 units, as determined by ImageJ analysis. After 300 seconds of exposure (5 minutes), the TFI was 144.51 units. This represented a 68% decrease in TFI.

Measurements of TFI showed that HeLa cells labeled with GAM IgG-iFluor^® 488 had an initial intensity of 1213.87 units, as determined by ImageJ analysis. After 300 seconds of exposure (5 minutes), the TFI was 885.29 units. This represented a 27% decrease in TFI.

 

Discussion

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One of the key motivators for this experiment was to determine if iFluor^® 488 had the potential to replace FITC as a green fluorescent dye for cell labeling applications. From this experiment, it was shown that iFluor^® 488 had much better photostability compared to FITC when conjugated to IgG and used to label tubulin. The fluorescence intensity loss of FITC was more than double that of iFluor^® 488 (68% vs 27%) over the same period of time. Thus, for applications which require photostable dyes or long exposure times, iFluor^® 488 is clearly superior.

Another quality to note is the dramatic difference in fluorescence intensity when comparing FITC labeled cells to iFluor^® 488 labeled cells. In captured images shown in Figure 2, iFluor^® 488 appears much brighter. This notion is supported by the intensity values, which demonstrates significantly greater intensity values for iFluor^® 488 than for FITC. This stark contrast was perhaps most evident in the image slice taken at 300 seconds, wherein the FITC labeled cells were barely visible whereas the iFluor^® 488 labeled cells were still clearly visible.

 

Conclusion

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In a direct comparison of FITC and iFluor^® 488, it was shown that iFluor^® 488 has a superior fluorescence intensity. Furthermore, iFluor^® 488 is significantly less susceptible to photobleaching, allowing for stained cells to be detectable for a much longer period of time. Based on these findings, iFluor^® 488 can serve as a superior alternative to FITC for cell labeling applications.

 

Additional Resources

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