title: Automated analysis of two- and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion.
abstract: The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events.
authors.affiliation: David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Rebhahn
authors.forename: Courtney
authors.initials: C
authors.lastname: Bishop
authors.forename: Anagha A
authors.initials: AA
authors.lastname: Divekar
authors.forename: Katty
authors.initials: K
authors.lastname: Jiminez-Garcia
authors.forename: James J
authors.initials: JJ
authors.lastname: Kobie
authors.forename: F Eun-Hyung
authors.initials: FE
authors.lastname: Lee
authors.forename: Genny M
authors.initials: GM
authors.lastname: Maupin
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Snyder-Cappione
authors.forename: Dietmar M
authors.initials: DM
authors.lastname: Zaiss
authors.forename: Tim R
authors.initials: TR
authors.lastname: Mosmann
mesh_terms: D000465:Algorithms; D000818:Animals; D001185:Artificial Intelligence; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010363:Pattern Recognition, Automated / Q000379:methods*
chemical_list: D016207:Cytokines