Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells.
OBJECTIVE
To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM).


METHODS
Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI.


RESULTS
FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained.


CONCLUSIONS
QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.
Kahn
Edmond
E
National Institute of Health and Medical Research, U678/Mixed Research Unit S UPMC, Paris, France. kahn@imed.jussieu.fr
Ménétrier
Franck
F
Vejux
Anne
A
Montange
Thomas
T
Dumas
Dominique
D
Riedinger
Jean-Marc
JM
Frouin
Frédérique
F
Tourneur
Yves
Y
Brau
Frédéric
F
Stoltz
Jean-François
JF
Lizard
Gérard
G
D000465:Algorithms; D018955:CD36 Antigens / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007653:Ketocholesterols / Q000494:pharmacology*; D036641:Microscopy, Fluorescence, Multiphoton; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D017785:Photons; D045663:Quantum Dots; D020298:U937 Cells
D018955:CD36 Antigens; D007653:Ketocholesterols; C003001:7-ketocholesterol