Dual Color Elispot
Dual Color Elispot - Related Videos
04:20
Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay
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2025
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.
11:53
ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes
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2023
Source: Tonya J. Webb1 1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in...
10:00
Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy
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Cited by 12 •
2014
Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.
08:26
The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity
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Cited by 10 •
2016
Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.
10:25
Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes
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Cited by 4 •
2024
This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.
FAQs
The two detection systems are central to distinguishing the measured secretions. Each system binds its corresponding target after cells release cytokines onto the capture-antibody-coated membrane, then produces a distinct colored signal. Using different colors allows the assay to display responses to both analytes in the same cellular measurement rather than collapsing them into one undifferentiated readout.
Spot color patterns add information beyond spot number. A spot associated with one color indicates secretion of one measured analyte, whereas a dual-color pattern identifies a cell response involving both. Thus, researchers can separate single-secreting and dual-secreting responses and assess cytokine multifunctionality within the responding population.
Unlike a cell-count measurement, this assay reports whether cells are functionally secreting measured cytokines after stimulation. The number of spots estimates the frequency of responding cells, while the color pattern adds information about secretion combinations. This distinction helps researchers compare immune activity, not merely the presence or abundance of cells.
The assay can be configured around antigen-stimulated cells or cells activated by another approach. This flexibility lets investigators examine pathogen-specific responses when an antigen is relevant, while also evaluating activation-dependent secretion more broadly. The resulting spot profile can then be compared across experimental groups to characterize differences in immune responsiveness.
A practical workflow starts by placing cells on a membrane coated with capture antibodies, followed by antigen stimulation or another activation condition. After secretion is captured, two target-specific detection systems generate distinct colors. Counting total and color-defined spots provides the frequency and secretion pattern needed for comparison between experimental samples.
Researchers apply the method to compare pathogen-specific T-cell responses, characterize multifunctionality, evaluate vaccines, and monitor immune responses during infection or treatment. Its combined frequency and color readouts help distinguish how many cells respond from whether they secrete one analyte or both, supporting functional comparisons across disease, intervention, or study groups.