• Multiplex Cytokine Measurement Analysis

    Multiplex Cytokine Measurement Analysis

    Posted on Jul 28, 2020

    Cytokines​ are a broad and loose category of small ​proteins​ ​(5-20 kDa molecular weight) ​important in ​cell signaling​, particularly in the regulation of immune system responses to infection and inflammation. Cytokines​ are ​peptides​ and cannot cross the ​lipid bilayer​ of cells to enter the ​cytoplasm​. They communicate by signaling through cell surface receptors. ​Although secreted, cytokines are not considered to be hormones. ​There are several families of cytokines expressed in humans and other vertebrates and can act powerfully as individual molecular reagents although most often exert their influence combinatorially in multiple interactions depending on the context in which they are expressed. ​The different types of cytokines, e.g., interferons, interleukins, lymphokines, chemokines, and tumor necrosis factors can act alone, work together, or work antagonistically in modulating a combinatorial response. Cytokines exert their influence on cells via cognate cell-surface receptors in either a paracrine fashion, i.e., by binding to receptors on other nearby cells, or in an autocrine mode, i.e., binding to receptors on the cell that secreted them.

    Multiplex Measurement of Cytokine Expression

    While familiar and reliable ELISA​ assays provide an excellent means of measuring the concentrations of individual cytokines, establishing expression profiles of multiple cytokines at once by multiplexing can be even more useful and informative during the development of a ​cell-based assay​. Multiplex measurements are technically challenging but can be accomplished by use of flow cytometers.

    Cytokine families and their characteristics

    Interferons (IFN)​ ​act by inhibiting viral replication. Infected cells produce and release interferons to alert uninfected cells to the presence of the virus.

    Interleukins (IL)​ were initially thought to be expressed only by leukocytes (white blood cells) but were later seen to be expressed by other cell types. Different members of the family have pro-inflammatory or anti-inflammatory properties with essential roles in the activation and differentiation of immune cells. Lymphokines​ are produced by lymphocytes, the subset of leukocytes that includes T cells, B cells, and natural killer (NK) cells. Typically produced by T cells to coordinate immune system responses, lymphokines have multiple functions, including the attraction of macrophages to an infected site. Lymphokines also participate in the production of antibodies by B cells.

    Chemokines ​are chemotactic cytokines. Their function is to act as a chemoattractant in guiding the migration of cells in following a signal of increasing chemokine concentration toward its source.

    Tumor necrosis factors (TNF)​ ​are transmembrane proteins expressed predominantly by immune cells that are released by extracellular enzymatic cleavage to function as cytokines in immune response and inflammation.

    Cytokine storms and Covid-19

    As part of the 2020 COVID-19 pandemic, there has been much mention of the lethality of so-called cytokine storms. Cytokine storms are particularly interesting because they can occur in young “healthier” patients that otherwise should recover better from SARS-Cov-2 infection. Cytokine storms represent a severe immune reaction in which too many cytokines are released into the blood too quickly. However, these are not necessarily just inflammatory cytokines, anti-inflammatory cytokines are also involved. Medical science continues to work to understand how to best treat these dangerous overproductions of cytokines that are often part of the body’s response to sepsis. Cytokine storms can occur not only as the result of an infection, but also because of autoimmune conditions or after some types of immunotherapy treatment. Some cytokines trigger apoptosis, i.e., programmed cell death, that can be a protective mechanism to prevent the spread of a disease. But if apoptosis begins to affect too many cells, significant tissue death can result – lung tissue in the case of COVID-19. Alveoli leak and fill with fluid, leading to pneumonia and dangerously low blood oxygen levels that can then result in organ failure elsewhere in the body.

    Cytokines as biomarkers

    Although cytokines are known to possess many diverse and important functions, they often serve as valuable biomarkers in research that may not be focused directly on their roles in either inflammation or immune responses. A biomarker is a molecule whose measurable levels can be informative as to the success of an experimental approach or therapeutic treatment because of previous observations that particular levels of that molecule could be associated with certain outcomes. In such a situation, it may not be important to know why the biomarker went up or down, only that quantitating its presence is helpful in interpreting the overall result of the experiment or treatment.

    MarinBio scientists are experts in molecular biomarker analysis​.

    In the case of cytokines, researchers may attempt to associate particular expression profiles of multiple biomarkers with various outcomes, especially when the measurement of the levels of individual cytokines, e.g., by ELISA, is not sufficiently instructive. For this reason, multiplex measurements of a large number of cytokines using more advanced technology is often desirable.

    Bead-based measurements of cytokines on flow cytometers

    One method for performing multiplex measurements of cytokines with flow cytometry​ uses antibodies to these proteins that have been conjugated to small fluorescent beads. Such beads can be analyzed using a flow cytometer, an instrument typically used to analyze fluorescently labeled cells. However, the fluidics, (excitation) lasers, and (emission) detectors of flow cytometers lend themselves to the use of bead-based assays. Beads of different sizes and stained with different levels of a fluorescent dye can be discriminated against by the instrument. Antibodies raised against a single cytokine (analyte) are conjugated to a particular bead type. These “capture” beads are incubated with a sample – typically serum, plasma, or cell culture media – to enable binding of the various analytes to their cognate antibodies. The beads are washed and then biotinylated “detection” antibodies against the analytes are added and allowed to bind. Finally, streptavidin conjugated to another fluorescent dye, phycoerythrin (PE), is added and binds tightly to the biotin molecules on the detection antibodies. The bound beads are run through a flow cytometer and the PE signal intensity for the different bead types, each representing a different cytokine, are quantitated for software analysis of the cytokine profiles of experimental samples versus controls.

    MarinBio can quantitate up to 40 different cytokines in a single sample using bead-based multiplex immunoassays.

    Example of Multiplex Cytokine assay Marin Biological Laboratories


    Multiplex Cytokine Assay results for PBMCs after 48 hours of drug incubation, showing cytokine concentrations in pg/mL across different drug concentrations in µM.
    Multiplex Cytokine Assay results showing cytokine concentrations (pg/mL) in PBMCs after 48 hours of drug incubation across varying drug concentrations (µM).


    Multiplex Cytokine Assay. Peripheral blood Mononuclear cells (PBMCs) were isolated and incubated with a drug​ for 48 hours. The mutiplex cytokine assay was used to analyze the amount of cytokine secreted into the supernatant media. Drug concentration (µM) versus cytokine concentration (pg/mL) is represented in the bar charts.