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High-Sensitivity Immunofluorescence Staining: A Comparison of the Liposome Procedure and the FASER Technique on mGR Detection

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Abstract

Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1–10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50–100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail.

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Acknowledgments

We would like to thank Manuela Krüger for providing the liposomes, Miltenyi Biotec for producing the anti-Dig/anti-Bio matrix and Timea Berki for providing the anti-GR antibody.

Authors Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published.

Competing Interest Statement

There are no competing interests.

Financial Support

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bu 1015/7-1) and from the Deutsche Sparkassenstiftung Medizin to FB. Contributions of Martin Hahne were made possible by DFG funding through the Berlin-Brandenburg School for Regenerative Therapies GSC 203.

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Authors and Affiliations

  1. Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charitéplatz 1, 10117, Berlin, Germany

    Cindy Strehl, Timo Gaber, Manuela Jakstadt, Martin Hahne, Paula Hoff, Cornelia M. Spies, Alexander Scheffold, Gerd-Rüdiger Burmester & Frank Buttgereit

  2. German Rheumatism Research Center (DRFZ), 10117, Berlin, Germany

    Cindy Strehl, Timo Gaber, Manuela Jakstadt, Martin Hahne, Paula Hoff, Alexander Scheffold & Frank Buttgereit

  3. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), 13353, Berlin, Germany

    Timo Gaber, Manuela Jakstadt & Frank Buttgereit

  4. Berlin-Brandenburg School of Regenerative Therapies (BSRT), 13353, Berlin, Germany

    Martin Hahne

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  1. Cindy Strehl
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Correspondence to Cindy Strehl.

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Strehl, C., Gaber, T., Jakstadt, M. et al. High-Sensitivity Immunofluorescence Staining: A Comparison of the Liposome Procedure and the FASER Technique on mGR Detection. J Fluoresc 23, 509–518 (2013). https://doi.org/10.1007/s10895-013-1163-4

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  • DOI: https://doi.org/10.1007/s10895-013-1163-4

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