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Volume 8, Issue 3 (Suppl) J Clin Cell Immunol, an open access journal
ISSN: 2155-9899 Euro Immunology 2017
June 29-July 01, 2017
June 29-July 01, 2017 Madrid, Spain
8th European
Immunology Conference
Development of flow cytometry based adherence assay for Nessieria gonorrhoeae using 5′-carboxy-
fluorosceinsuccidyl ester (CFSE) and ME-180 cells
SD Thakur, M Obradovic, JR Dillon and HL Wilson
University of Saskatchewan, Canada
Statement of the Problem: The microorganism Nessieria gonorrhoeae is an obligate human pathogen and its adherence to host cells is
essential for its pathogenesis. We devised a flow cytometry-based method to quantify the adherence of piliated N. gonorrhoeae strain
F62 to human cervical ME-180 cells.
Methodology: Piliated N. gonorrhoeae F62 were collected after 10 to 12 hours of growth then stained with cell-permeable fluorescent
dye 5'-carboxyfluoroscein succidyl ester (CFSE). The bacteria were incubated with 0.5 μl of 5 mMCFSE in 2.5 ml of PBS and incubated
at 37°C for 15 min. ME-180 cells were incubated for 2 hours with fluorescent, piliated N. gonorrhoeae (multiplicity of the infection
1:100) then the ME-180 cells were washed with phosphate buffer saline to remove loosely adherent bacteria. Flow cytometry was used
to quantify the percentage of ME-180 associated with CFSE+ fluorescent bacteria and a minimum of 30,000 events were recorded.
Finding: Results indicated that 19.2% ± 0.99 (n=4) ME-180 cells were associated with the fluorescent, piliated bacteria. To assess
whether antibodies specific for N. gonorrhoeae blocked their adherence to ME-180 cells, rabbit hyper-immune anti serum was raised
against heat-killed piliated N. gonorrhoeae F62. Adherence efficiency, the percentage of cell-associated CFSE+ bacteria divided by the
total input CFSE+ bacteria ranged between 37-47% (n=5). Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly
inhibited gonococcal adherence by 6 and 3 fold, respectively. Heat-inactivated negative rabbit serum was significantly (3 to 5 folds)
less effective at preventing bacterial adherence suggesting that antibody specificity and not a non-specific serum component were
involved. Flow cytometric analysis was amenable to the quick, easy and high-throughput quantification of N. gonorroheae association
with eukaryotic cells. These approaches may be adapted for use in in vitro and in vivo adherence studies related to gonococcal
pathogenesis.
SD Thakur et al., J Clin Cell Immunol 2017, 8:3(Suppl)
DOI: 10.4172/2155-9899-C1-037