title: High-sensitivity immunofluorescence staining: a comparison of the liposome procedure and the FASER technique on mGR detection.
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.
authors: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany. strehl@drfz.de
authors: Cindy
authors: C
authors: Strehl
authors: Timo
authors: T
authors: Gaber
authors: Manuela
authors: M
authors: Jakstadt
authors: Martin
authors: M
authors: Hahne
authors: Paula
authors: P
authors: Hoff
authors: Cornelia M
authors: CM
authors: Spies
authors: Alexander
authors: A
authors: Scheffold
authors: Gerd-Rüdiger
authors: GR
authors: Burmester
authors: Frank
authors: F
authors: Buttgereit
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008081:Liposomes / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008081:Liposomes; D011965:Receptors, Glucocorticoid