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Flow cytometric analysis of intracellular interferon-gamma synthesis in rat CD4 T cells
Insights
Researchers developed a new method to analyze single rat T cells for cytokine production. This technique successfully stains for interferon-gamma (IFN-γ), a key Th1 cytokine, enabling better understanding of T cell responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Current methods for analyzing cytokine expression in rat T cells lack single-cell resolution for simultaneous cytokine production.
- Intracellular cytokine staining via flow cytometry offers single-cell analysis, but requires specific antibodies.
- While antibodies for Th2 cytokines (IL-4, IL-10) are available for rat T cells, Th1 cytokine antibodies are lacking.
Purpose of the Study:
- To establish a reliable method for intracytoplasmic cytokine staining of interferon-gamma (IFN-γ) in rat CD4 T cells.
- To validate the specificity and effectiveness of a novel monoclonal antibody (DB1) for IFN-γ detection.
- To correlate intracellular IFN-γ staining with secreted IFN-γ levels measured by ELISA.
Main Methods:
- Utilized flow cytometry for intracellular cytokine staining.
- Employed a mouse anti-rat IFN-γ monoclonal antibody (DB1) for intracytoplasmic staining.
- Confirmed antibody specificity using unlabeled antibody excess and recombinant IFN-γ.
- Correlated intracellular staining results with ELISA analysis of supernatant cytokine levels.
Main Results:
- The DB1 antibody was successfully used for intracytoplasmic staining of IFN-γ producing rat CD4 T cells.
- Specificity of the DB1 antibody for IFN-γ staining was confirmed through blocking experiments.
- Intracellular IFN-γ staining results showed a strong correlation with IFN-γ production measured in culture supernatants via ELISA.
Conclusions:
- A validated method for intracellular IFN-γ staining in rat CD4 T cells using the DB1 antibody is presented.
- This technique overcomes previous limitations in analyzing Th1 cytokine production at the single-cell level in rats.
- The findings enable more comprehensive characterization of T cell responses involving IFN-γ production.
Abstract:
To date the techniques used to analyse cytokine expression by rat T cells do not give information about the simultaneous production of different cytokines from individual cells. Recently, a method for analysing the intracellular production of cytokines at the single cell level using flow cytometry has been developed. It is well established that the most critical requirement for successful intracellular cytokine staining is the availability of appropriate antibodies. In rat, it is possible to stain for intracellular IL-4 and IL-10 (Th2 cytokines) using the commercially available antibodies but not for Th1 cytokines. In the present work, we show that DB1, a mouse anti-rat IFN-gamma monoclonal antibody, could be used for intracytoplasmic staining of IFN-gamma producing rat CD4 T cells. The specificity of the staining was confirmed using a molar excess of unlabelled antibodies or recombinant cytokine. Finally, intracellular staining for IFN-gamma correlates with cytokine production in culture supernatant as evaluated by ELISA analysis.
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