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Measuring cell-mediated immunity (CMI) by Flow Cytometry

Flow Cytometry is considered the method of choice to measure Cell-Mediated Immunity (CMI) by assessing the level of intracellular cytokines after antigen exposure.

CMI is a branch of the adaptive immune response that allows protection against various pathogens. This protection is mainly carried out by the activation of T-cells in response to an infection. Assessing CMI is critical to determine the efficacy and long-term effects of new vaccines.

Crux Biolabs offers multiple platforms to measure CMI, including Flow Cytometry and ELISpot. Intracellular Cytokine Staining (ICS) by Flow Cytometry is the method of choice to measure specific T-cell responses to vaccines or pathogens. Antigen-stimulated Peripheral Blood Mononuclear Cells (PBMCs) are analysed by Flow Cytometry to measure multiple intracellular markers simultaneously at the single cell level. Combined with phenotyping markers, this method provides a quantitative analysis of the phenotypic and functional differences within multiple subtypes of responder cells.

Patient blood samples are received fresh and PBMCs are isolated using a density gradient medium and cryopreserved in liquid nitrogen before further analysis. Frozen PBMCs are thawed and stimulated with the antigens of interest. These antigens will be processed by antigen-presenting cells, such as monocytes, and then be presented to lymphocytes. Responsive lymphocytes will consequently express various cytokines, that can be stained using appropriate labelled antibodies and detected by Flow Cytometry.

With Crux Biolabs’ suite of Flow Cytometers, we can assess up to 21 parameters to provide answers to your CMI requirements. A basic and routinely used panel would include expression of TNF-α, IFN-γ and IL-2 within CD4 and CD8 T-cell populations. Additional markers to measure the potential of CD8+ T-cells to mediate killing of target cells, such as CD107a, granzymes or perforins can easily be included.

To assure consistency between patient timepoints, Crux Biolabs offers sample storage services before undertaking Flow Cytometry as a batch process. This more efficient approach reduces project costs.

Cells in blue have not been stimulated with any antigens and do not express TNF-α or IFN-γ. After stimulation with viral antigens (in orange), cells have been activated, express TNF-α or IFN-γ and support an immune response against the pathogen.

Crux Biolabs – internationally accredited in Flow Cytometry

With our strong commitment to providing the best results to clients, Crux Biolabs is proud to participate in global quality assurance programs, ensuring our bioanalytical techniques meet international standards.

Crux Biolabs recently completed the EQAPOL Flow Cytometry Intracellular Cytokine Staining External Proficiency program, administered by Duke Human Vaccine Institute’s Immunology and Virology Quality Assessment Center. Our team of expert scientists achieved a score of 95.5 out of 100, placing Crux in the highest category of ‘Excellent’ laboratory.