6 well plate

Live-cell imaging
of the immune system
in health and disease

  • Immunology

The immune system is a highly complex biological network of organs, cells, and molecules that protect the host against infectious diseases and even some forms of cancer. The intrinsic complexity of the immune system and its involvement in a multitude of fundamental physiological processes, demands the application of a wide range of scientific tools that can provide an in-depth insight into immune function. The field of immunology relies heavily on traditional in vitro techniques, including ELISA, immunoblotting, and flow cytometry. However, these approaches give limited information about transient cell-cell interactions, localization of immune cells, and their morphology.

Time-lapse imaging of cell cultures is a powerful approach that can shed light on fundamental aspects of immune cell behavior by visualizing complex biological processes in real time, in physiologically relevant conditions. Live-cell imaging allows scientists not only to address unresolved research questions, but also to save time and resources while doing so.

Cell culture in incubator
Labelled and label-free monitoring of immune cell proliferation

T-cell number and function can be examined via assays that detect T cell activity, including T-cell proliferation, cytokine production, and cytotoxicity. During infection, antigen-specific T lymphocytes undergo robust proliferation to form a large pool of effector T cells. This ability of T cells to proliferate in response to an antigen is measured by a lymphoproliferation assay and it has been used to indicate the presence of antigen-specific CD4+ helper T cells in a sample. To perform this assay, tested peripheral blood mononuclear cells (PBMCs) or purified T cells are mixed with the antigen, and the proliferation of cells is assessed using an endpoint assay. In contrast, live-cell imaging allows to monitor and quantify immune cell proliferation for hours or even days at a time, without missing any critical time points. Depending on the experimental set-up, both labelled and label-free approaches can be used to visualize and measure immune cell proliferation.

Determining immune cell numbers and viability

Human peripheral blood mononuclear cells (PBMCs) are blood cells that contain a round nucleus, such as lymphocytes, monocytes, and macrophages. Constituting a major part of the immune system, PBMCs play a key role in cell-mediated and humoral immune responses against invading pathogens. Following isolation of PBMCs from whole blood, the concentration of these cells is measured, which can be a challenging and time-consuming process. The CytoSMART Exact FL fluorescence cell counter is optimized for working with PBMCs and other hard-to-detect cell types. Together with appropriate fluorescent probes (e.g. AO/PI), the counter accurately recognizes nucleated cells in a sample, while excluding red blood cells, platelets, and other cellular debris from the count. In addition, the AO/PI staining can be used for assessing the viability of PBMCs.

Cyto SMART Lux3 FL fluorescence
Visualization of dynamic immune responses

Phagocytosis is a key mechanism of the innate immune response that is used by neutrophils, macrophages, and other professional phagocytes to remove pathogens and cell debris from the body. To visualize and measure the process of phagocytosis, foreign material is tagged using pH-sensitive fluorescent probes that emit a signal upon exposure to the acidic environment of the phagosome. The CytoSMART Lux3 FL is a portable fluorescence live-cell imager that can track dynamic cellular processes, such as phagocytosis, in real time and under optimal cell culture conditions. In addition, the new Object Count algorithm of the CytoSMART Lux3 FL can automatically calculate the number of fluorescent objects in the image and investigate how this number changes over the course of time.

Real-time analysis of immune cell chemotaxis

The ability of certain immune cells, such as neutrophils and T cells, to detect an extracellular chemical signal and migrate in response to it, plays a crucial role in immune system function and homeostasis. The analysis of cell migration requires the application of high-throughput methods that can visualize and quantify cells’ motility in space and time. The CytoSMART live-cell imaging platforms, including the CytoSMART Omni and Lux3 FL, can be used for direct visualization and tracking of both, single-cell (e.g. macrophages, B and T lymphocytes) and collective cell migration (e.g. tumor cells). In case of single cell migration, the recorded time-lapse videos can be analyzed using commercially available (MetaMorph by BioImaging Solutions, Inc.) or free (TrackMate by ImageJ) software packages. Collective cell migration can be investigated using a wound-healing assay, with the changes in the rate and efficiency of cell movement quantified by means of the integrated CytoSMART image analysis software.

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