Candida Infection Biology – fungal armoury, battlefields ... - FINSysB

Poster number: 30
A Novel Flow Cytometric Protocol for Assessment of Yeast
Cell Adhesion
A. Silva-Dias 1,2 , I.M. Miranda 1,2 , R. Rocha 1 , M. Monteiro-Soares 3 ,
A. G. Rodrigues 1,2,4 , C. Pina-Vaz 1,2,5
1 2 Department of Microbiology, Faculty of Medicine, University of Porto, Cardiovascular
Research & Development Unit, Faculty of Medicine, University of Porto; 3CINTESIS, Department of Biostatistics and Medical Informatics, Faculty of Medicine, University of Porto,
4 5
Burn Unit and Department of Plastic and Reconstructive Surgery, Hospital S. João,
Department of Microbiology, Hospital S. João, Porto, Portugal.
The capacity of microorganisms to adhere to each other, to biomaterials, to biotic
substrates and tissues is an important trait at environmental, industrial or medical
level. Pathogenic yeasts belonging to genus Candida are frequently involved in
systemic infections, mainly due to its ability to adhere and colonize tissues and
medical indwelling devices. In order to quantify this attribute, several methods have
been used, with advantages and drawbacks. Some of the major limitations found
within these methodologies concern the incubation time, the small number of cells
analyzed and the operator`s subjectivity. In order to overcome these aspects we
have developed a quantitative method to measure yeast cells` adhesion through
flow cytometry. The adhesion assay is based upon a simple principle: yeast cells
became fluorescent when attached to highly green fluorescent microspheres.
Therefore by flow cytometry a quantitative distinction between non-adherent yeast
cells (non fluorescent) and adherent cells (fluorescent) is achieved. By measuring
the intensity of fluorescence emitted by each cell it is possible to evaluate the
number of beads that are attached to each cell. Additionally, fluorescent beads
could be coated with different substrates to which yeast adhesion ability can be
tested.
A suspension of yeast cells (1x10 6 cell.ml -1 ) is mixed with green fluorescent
polystyrene microspheres, uncoated or coated with host proteins, and incubated
for 30 minutes. Samples (50.000 cells) are analyzed in the FL3 fluorescence channel
(FACSCalibur BD Biosciences). Within two hours an adhesion profile is obtained
based on three parameters: percentage of adherent cells, mean of fluorescence
intensity (MFI) and cells-microsphere population`s distribution pattern. Comparing
to the classical assays, flow cytometry protocol represents a useful tool to quantify
yeast adhesion to different substrata in a large scale, providing manifold data in a
speedy and informative manner.
A Silva-Dias is supported by a FCT (Fundação Ciência e Tecnologia) doctoral grant
SFRH/BD/44896/2008; I Miranda is supported by Ciência 2008 (FCT) and European Social Fund.
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