Abstract 5473: Development of a novel fluorescent ELISpot assay using the CeligoTMImage Cytometer

ELISpot and its variant, fluorescent ELISpot (FluoroSpot), are commonly used methodologies that monitor and detect cytokine and/or antibody secretion from peripheral blood mononuclear cells (PBMCs). Conventional ELISpot and FluoroSpot assays utilize PVDF-membrane plates, which require labor-intensive washing steps that may introduce variability in performance and results. Here, we have developed a novel FluoroSpot assay using polystyrene-, clear-bottom microplates with simplified automated washing steps to minimize operator variability and error. In this work, we assessed multiple critical cellular immune-related activities, including IL-6, IFN-g, and TNF-a secretion in PBMCs using this novel FluoroSpot methodology in comparison to the conventional ELISpot assay. To detect cytokine secretion of IL-6, IFN-g, and TNF-a, the Celigo™ Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA) was incorporated to capture whole-well images of fluorescently labeled detection antibodies. Results for both methodologies were comparable to one another, validating the use of polystyrene plates on Celigo™ as opposed to PVDF-membrane plates. In addition to FluoroSpot detection, the Celigo™ was used as a quality control check to provide a comprehensive analysis of live cell distribution prior to performing the FluoroSpot assay. Live cell imaging results were used to correlate downstream cytokine-releasing cell spot formation results. Combinational use of the novel FluoroSpot polystyrene plate with the Celigo™ reduced operator variability and errors, while streamlining a multiparametric analysis to identify the number of fluorescent spots, spot morphology (size), and whole-well mean fluorescence intensity values. This methodology may prove useful for researchers interested in monitoring and detecting cytokine and/or antibody-releasing PBMCs. Citation Format: Wen-Chieh Liao, Julia Abdalla, Carolina Franco Nitta, Mackenzie Pierce, Bo Lin, Jessie Ni. Development of a novel fluorescent ELISpot assay using the CeligoTMImage Cytometer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5473.