Development of flow cytometry based adherence assay for Nessieria | 55560
Journal of Clinical and Cellular Immunology

Journal of Clinical and Cellular Immunology
Open Access

ISSN: 2155-9899

Development of flow cytometry based adherence assay for Nessieria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester (CFSE) and ME-180 cells

8th European Immunology Conference

June 29-July 01, 2017 Madrid, Spain

SD Thakur, M Obradovic, JR Dillon and HL Wilson

University of Saskatchewan, Canada

Posters & Accepted Abstracts: J Clin Cell Immunol

Abstract :

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 ?¼l of 5 mM CFSE 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.

Biography :