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Focus Labo : Anti-Glycophorin Specificity In Human Rbc Agglutination Characterized By Flim-Fret Microscopy. Dr Dominique DUMAS1, Pr Bibiana D. RIQUELME 2, Natalia de Isla1. 1 Département d’Imagerie et de Biophysique Cellulaire. Laboratoire Lemta UMR CNRS 7563 et IFR CNRS 111 « Mécanique et d’Ingénierie Cellulaire et Tissulaire ». 54505 Vandoeuvre-les-Nancy, France. Tél : (33)03 83 68 34 64 – email@example.com. 2. Facultad Cs. Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario. ARGENTINA. Short description of our department The Laboratory of Cell and Tissue Engineering The “Imaging and Biophysic” department of the created by Pr Stoltz in 1996 is focused on the group uses some advanced cytometric and mechanobiology of cells and tissues. The primary spectroscopic techniques to detect biological concern of our research group (UMR CNRS 7563 events involved in these interactions including and IFR CNRS 111) is to observe and understand flow cytometry, optical scanning the physiological consequences of applied microscopy/deconvolution, multiphoton mechanical stresses in vascular, blood and microscopy and fluorescence lifetime imaging cartilage cells. microscopy (FLIM). Objective In this short study, we considered the FRET signal Cells (RBC) by specific monoclonal antibodies to characterize the agglutination of Red Blood (anti-glycophorin A or B). Introduction Glycophorins A, B and C are abundant in RBC. Different approaches of FRET have been red blood cell (RBC) transmembrane integral developed to characterize the hemoagglutination proteins. Their highly glycosylated nature and high (Figure 1) based on the interaction of fluorophores sialic acid content account for the net negative (Alexa488TM, DiO and DiI) located in the RBC charge of mature RBC membrane, which is membrane or combined to secondary antibody physiologically important because it impedes any directed against the primary agglutining Mab. tendency to stick together in the circulation. Combined intensity (spectral) and Lifetime (FLIM) Glycophorin A (GPA) is specific to RBC and is Imaging was used to discriminate the FRET signal found at high density on the extra-cellular surface, of molecules on their different lifetimes whereas possibly linked also to Band3. In this study, we their emission spectra overlap (Alexa488TM or DIO have tested anti-(GPA plus GPB) specific mouse / DiI) as independent phenomena of the monoclonal antibodies (agglutining Mab) as fluorophore concentration and photobleaching. primary antibody (Table I) to directly agglutinate Methods For the image series and spectra Imaging module (Becker&Hickl, Berlin) was measurement, a Leica TCS SP2-AOBS equipped interfaced (signals, Pixel Clock, Frame Sync) to with an acousto-optical beamsplitter and Argon the scan controller of the Leica TCS-AOBS (457nm, 476nm, 488nm, 514nm) and HeNe Multiphoton laser scanning microscope. The (543nm and 633nm) lasers were used with a decay analysis measured by time-correlated x63/1,32 oil immersion. Fluorescence lifetime single photon counting was performed using the measurements were performed with a time- SPCImage software (Becker&Hickl GmbH). The correlated single photon counting technique using actual time resolution depends on the detector a Ti-sapphire laser pumped by a continuous wave with the PMT typically built-in the microscope argon laser (Mira 900F-Verdi 8W, Coherent, attached to the non-descanned port (high pinhole fully opened, beam expander 3) with a sensitivity photomultiplier R6357 Hamamatsu with pulse width from 120 fs and a repetition rate of 76 a typical rise time of 1.4 ns). MHz. For lifetime imaging, SPC-730 TCSPC GR GR G G G G R R R R TM Alexa-488 TM Alexa-488 A B C Figure 1. Confocal images of Red Blood Cells. 1A. For control, RBC agglutination has been verified in the presence of agglutining antibody and revealed with a secondary antibody combined to Alexa488 TM. 1B. Two RBC populations have been separately labeled with 1-1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiO, 5.7 mM, Molecular Probes) or 3,3’ – dioctadecyloxacarbocyanine perchlorate (DiI, 2.15 mM, Molecular Probes) and agglutination of a mixture of both population has been made in the presence of agglutining Mab. 1C. The DiI-RBC were agglutinated in the presence of Mab and revealed by secondary antibody combined to Alexa488 TM. This is a donor (Alexa-488TM)- acceptor (DiI) couple in a Forster resonance energy transfer process (FRET), whereby the fluorescence of the donor is quenched by the acceptor whose fluorescence is enhanced provided these fluorophores are in close proximity and the fluorescence of the donor overlaps the absorption spectrum of the acceptor. Then 33 µL of the stock solution were added to RBC. In agreement with the low absorbance of DiI at 488 nm ( 8000 M-1 cm-1) as compared to that of Alexa-488TM ( 65000 M-1 cm-1), their similar quantum yield of fluorescence and concentration ratio ([Alexa488TM]= 20[DiI]), the contribution of the DiI fluorescence at 565 nm upon excitation at 488 nm is only due to the FRET process. Results All the antibody used was found to directly fluorescence alone from donor (without acceptor agglutinate the human RBC. In view of the excitation) or acceptor (without detection of fluorescence properties depicted in RBC for the fluorescence from unpaired donor). To avoid pair of fluorophore Alexa-488TM (donor) and DiI these limitations in the case of multi-labelling (acceptor), it may be expected that upon experiments, FLIM in dynamic-state provides a agglutination of RBC, effective FRET should be discrimination of molecules in their fluorescence observed. FRET is a distance-dependent lifetime, which allows to evaluate the underlying interaction between electronic states and depends mechanism of energy transfer process. Figure 3 on the inverse sixth power of the intermolecular demonstrates upon excitation at 780 nm the interaction distance. For 3-17 Mab (Anti-GPA), the FLIM-FRET from Alexa488TM to DiI for the 3-16 proximity requirement for the occurrence of FRET and 3-46 Mabs only with a lifetime distribution in cannot be met upon excitation at 488 nm as the picosecond range. Similarly, effective FRET shown in Figure 2 which displays the fluorescence was not observed for the 3-17 and 3-47 Mabs. spectrum (without acceptor emission at 565 nm). The contrast in measured lifetime image (Figure The FRET from Alexa-488TM to DiI was found for 4) is a reliable indicator for spatial variations in 3-16 Mab with detection of fluorescence from donor-acceptor association. All together, these acceptor. For 3-46 and 3-47 Mab, the spectra results only revealed by FRET-FLIM strongly analysis cannot solve the problems of spectral support the importance of the specific reactivity contaminations that arise in steady-state with Glycophorin A or B of agglutining Mab. (intensity) measurements, as specific detection of Figure 2 : Fluorescence spectra of RBC stained with DiI, then agglutinated with different anti-glycophorin A (3-17 and 3-47) or anti-glycophorin B (3-16 and 3-46) antibodies and revealed by secondary IgG antibody combined to Alexa 488TM. Excitation wavelength was respectively 488 nm for FRET in all the cases and adjusts to minimize the excitation of the acceptor. Fluorescence spectra were recorded in selected region on the RBC agglutinate to show different profiles (in and out the closed contact on the membrane of two RBC face to face). Figure 3: FRET image of RBC agglutinated with 3-16 Mab. The color is determined by the amplitude of the fluorescence lifetime values from 0 to 3 ns. Acknowledgement The authors would like to thank Dr. Blanchard (EFS Nantes - France) for the anti-GPA and anti-GPB specific mouse monoclonal antibodies supply. 700 600 500 3-16 400 3-46 UA 300 3-47 3-17 200 100 0 0 500 1000 1500 2000 2500 ps Figure 4 : Temporal histogram depicting the Alexa-488TM fluorescence lifetime distribution obtained by a biexponential fit of the fluorescence decay at each position in an image of RBC aggregate. We assume that the average lifetime measured is the sum of the true lifetimes of the donor in bound and unbound state, weighted by their respective population. The average lifetime of the alone fluorophore (Alexa488 TM) is around of 4,13 ns. When the Alexa488TM fluorophore is combined to secondary Mab (IgG), its average fluorescence lifetime is in the nanosecond range (1,7-2,2 ns) but upon transferring energy, it may drop in picoseconds (350-750 ps). The reduced lifetime of Alexa-488TM (indirectly combined to 3-16 and 3-46 Mab) indicates a molecular interaction with DiI incorporated in the RBC membrane. The proximity requirement for the occurrence of FRET was not met for the 3-47 and 3-17 Mab. References Wouters, F.S et al. (2001) Imaging biochemistry inside cells. TRENDS in Cell Biology.11, 203-211. Elangovan, M et al. (2003) Characterization of one-and two-photon excitation fluorescence resonance energy transfer microscopy. Methods. 29, 58-73. Jakobs, S et al. (2000) EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two photon and fluorescence lifetime microscopy. FEBS Letters. 479, 131-135.