Step-by-step guide to FluoroSpot

Published: September 7, 2023

Updated: June 19, 2024

FluoroSpot is the multiplex version of ELISpot. The assay combines the sensitivity of ELISpot with the capacity to study secretion of several analytes simultaneously. This is an in-depth guide for each step of the FluoroSpot protocol, including tips and tricks, key details to think about, and best practice.

FluoroSpot is an immunoassay used to quantify analyte-secreting cells. Just like in the ELISpot assay, target proteins secreted by cells are captured by specific antibodies immediately after secretion and throughout the stimulation process, making it an extremely sensitive sandwich assay. The difference to ELISpot lies in the mode of detection: where ELISpot relies on an enzymatic reaction, FluoroSpot utilizes fluorescence. This opens up for multiplex analyses of several analytes simultaneously, enabling studies of cell populations with different functional profiles.

We developed the technique in 2008 and have continually launched new kits and refined the protocol. Thus, we can offer many tips to fine-tune and maximize the success of your assay. We will discuss such suggestions in this guide, starting where the assay begins – with coating the FluoroSpot plate – and continue through the steps of the assay.

The general steps of a FluoroSpot assay

FluoroSpot assay principle

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