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Journal of Histochemistry and Cytochemistry, Vol. 45, 1307-1314, Copyright © 1997 by The Histochemical Society, Inc.

TECHNICAL NOTE

Detection of the Receptor for the Human Urokinase-type Plasminogen Activator Using Fluoresceinated uPA

Rosalba Ciccocioppoa, Maria G. Capria, and Saverio Albertia
a Laboratory of Experimental Oncology, Department of Cellular Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri-Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy

Correspondence to: Saverio Alberti, Institute Mario Negri Sud, 66030 S., Maria Imbaro (Chieti), Italy.

The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 x 105 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One µg of FITC-uPA efficiently stains 2 x 105 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells. (J Histochem Cytochem 45:1307-1313, 1997)

Key Words: urokinase-type plasminogen activator, fluorescein labeling, cell surface receptors, fluorescence microscopy, flow cytometry


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