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Bio-imaging method for cellular protein uptake

Cellular uptake and trafficking of matrix proteins, such as enamel matrix derived proteins, was demonstrated after tagging the protein with a fluorescent marker FITC (fluorescein isothiocyanate), adding it to human periodontal ligament cells in cell medium, and following the fluorescence with scanning laser confocal microscopy.

The fluorescein labelled material appeared to form discrete intracellular vesicles that seemed to form the classic endocytotic pathway. Fluorescein labelled material was found to co-localise following immunoprobing with rhodamine linked anti-amelogenin antibodies. The results indicate that amelogenins may be taken up by human periodontal fibroblasts and are subject to intracellular trafficking. In particular the 20KDa amelogenin appears to be taken up and degraded with time to generate 5KDa fragments by intracellular processing. This is a finding which indicates that the amelogenin polypeptides, such as TRAP, man impact cell biology in a way not previously known.

LABELLING METHOD: EMD was dissolved at 4 mg/ml sodium bicarbonate buffer pH 9. FITC in anhydrous DMSO (1 mg/ml) was slowly added to the EMD solution under gentle stirring (50 microliter of FITC solution per ml). The mixture was incubated in the dark at 4C for 5-8 hours, then ammonium chloride was added, and the reacion was incubated for a further 2 hours. Unreacted FITC was then removed by dialysis against PBS.

UPTAKE TESTING: Cells were grown to confluence in conjugate free culture medium on sterile glass coverslips placed in 6-well culture flasks in 5% carbondioxide in air at 37C. EMD-FITC was added to the culture medium (1:8, vol:vol)for varous time periods. Controls were similarly incubated with FITC labeled BSA. Following incubation with the labelled protein, the cells were washed with 0.1M formic acid pH2.2, 0.69% NaCl to remove extracellular precipitated EMD, followed by a PBS wash prior to fixing in formaldehyde ans washed in PBS. Where nuclear counter staining was required, cells were then permeabilised with 0.1% Triton X-100 washed in PBS and labeled with 0.1%TO-PRO-3 blue nuclear stain The cells were observed using confocal microscopy (FITC absorption and fluorescence emission maxima; 496nm and 520nm respectively; excitation light source; Green AR/ARKr laser setting 488nm. TO-PRO-3 absorption and fluorescence emission maxima: 642 and 661 nm respectively; excitation light source: Green HeNe laser setting 488 nm).

Reported by

University of Oslo
Faculty of Dentistry, PO Box 1109, Blindern
N-0316 Oslo
Norway
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