Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy.
OBJECTIVE
To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM).


METHODS
U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei.


RESULTS
FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained.


CONCLUSIONS
QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.
Kahn
Edmond
E
Institut National de la Santé et de la Recherche Médicale U678/Unité Mixte de Recherche-S Université Pierre et Marie Curie, Institut Fédératif de Recherche 14, Centre Hospitalier Universitaire Pitié-Salpêtrière, Paris, France. edmond.kahn@imed.jussieu.fr
Vejux
Anne
A
Ménétrier
Franck
F
Maiza
Christophe
C
Hammann
Arlette
A
Sequeira-Le Grand
Anabelle
A
Frouin
Frédérique
F
Tourneur
Yves
Y
Brau
Frédéric
F
Riedinger
Jean-Marc
JM
Steinmetz
Eric
E
Todd-Pokropek
Andrew
A
Lizard
Gérard
G
D001158:Arteries / Q000033:anatomy & histology* / Q000473:pathology; D050197:Atherosclerosis / Q000378:metabolism*; D018955:CD36 Antigens / Q000378:metabolism*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal / Q000379:methods; D009000:Monocytes / Q000378:metabolism*; D010799:Phycoerythrin / Q000737:chemistry; D045663:Quantum Dots; D020298:U937 Cells
D018955:CD36 Antigens; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate