The FluoroSpot assay is very similar to the ELISpot assay. The main difference is that the FluoroSpot assay is able to analyze the presence of multiple analytes on one plate of wells, whereas the ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay accomplishes this by using fluorescence rather than an enzymatic reaction for detection. The steps for a FluoroSpot assay are also similar, with a few differences.
# Antibody Coating: Similar to the ELISpot, cytokine-specific monoclonal capture antibodies are added to a plate with wells. For both assays, the plates are ethanol-treated to avoid contamination and skewed data collection. For the FluoroSpot assay, a mixture of different types of capture antibodies are attached to the wells in order to detect multiple types of analytes.  In order to get optimal results with the ELISpot and the FluoroSpot assay, proper plate coating techniques should be followed. The plates should be treated with ethanol, washed, and then coated with antibodies. Ethanol treatment methods also vary depending on the type of plates that are used. For MSIP and IPFL plates, you should add 15 micro liters of 35% ethanol to all of the wells. Allow the ethanol to sit in the wells for one minute, and then pour it out. For MAIPSWU plates, you should instead add 50 micro liters of 70% ethanol to all of the wells. Allow the ethanol to sit in the wells for two minutes, and then pour it out. After you have treated the wells with ethanol, you need to wash all of the wells with 200 micro liters of sterile water. This washing process should be repeated for a total of 5 times. Once the wells have been treated with ethanol and washed, the cytokine-specific monoclonal capture antibodies can be added to each well.
# Cell Incubation: Cell are added to the wells and are incubated in the presence or absence of stimuli that affect protein secretion.
# Cytokine Capture: Proteins/analytes that are secreted by the incubated cells will bind to the capture antibodies attached to the wells during the first step.
# Detection Antibodies: Similar to the ELISpot, once the wells are rinsed to get rid of the cells and other substances that we are not interested in identifying or measuring, a biotinylated detection antibody is added (this is specific for one type of analyte that you wish to quantify) and then tag-labeled detection antibodies are added for the second and third types of analytes being studied.
# Fluorophore-labeled Conjugates: Instead of adding a streptavidin-enzyme conjugate, the detection of multiple analytes is amplified in the FluoroSpot with the use of fluorophore-labeled anti-tag antibody and streptavidin-fluorophore conjugate. A fluorescence enhancer solution is also added during this step in order to enhance the signals later used when analyzing the fluorescence colors in the wells. This fluorescence is what makes it possible for the FluoroSpot to analyze and compare multiple analytes, unlike the ELISpot.
# Analysis: Since the FluoroSpot relies on the use of fluorescence and not an enzymatic reaction, there is no need for a step that adds a substrate to react with enzymes (as needed for the ELISpot). The last step for the FluoroSpot assay is to analyze the fluorophores under an automated fluorescence reader that has separate filters for the different fluorophores being analyzed. These filters should be selected for the specific wavelengths of the fluorophores if you want accurate measurements.
Since the FluoroSpot assay identifies and quantifies the presence of multiple analytes, it is possible that the absorption of one analyte can affect the secretion of another analyte; this is called capture effects. The effect an analyte has on another analyte could be positive or negative (the production of the second analyte can either increase or decrease). To counteract capture effects, it is possible to use co-stimulation in order to bypass the decreased production of an analyte. This is when a second antibody that stimulates the production of the same analyte is added to the wells.