Immunology research
Live-cell imaging
of the immune system
in health and disease
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.
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.
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.
Videos
Appnotes
Cell Culture Monitoring - Lux2
Cell Motility Video Monitoring - Lux2
Cell Proliferation
In general monitoring cell growth is performed manually, by quickly investigating the confluency of the cell culture using a bright-field microscope. Investigation of confluency provides insight in the cell culture growth rate and cell viability.
The study performed here automated live-cell imaging is investigated as an alternative to manual inspection of confluency. A 96-well plate was seeded with rat brain glioma cells (C6) at varying densities and monitored over a 2-day period. The imaging process was performed inside a standard CO2-incubator for the entire experiment, ensuring optimal cell culture conditions.
Cell Culture Monitoring - Lux2
Setting up cell cultures is easy enough. However monitoring your cultures and optimization is time-consuming and cumbersome. Waiting for the ideal confluency, quickly studying effects of various media means taking your cells in and out of the incubator more often than you would like.
Visualizing cell cultures from inside an incubator using a compact microscope that facilitates live cell imaging can overcome these issues. While live cell imaging has been restricted to costly, high-end devices, the CytoSMART Lux2 offers an affordable and easy-to-use alternative for virtually any lab. The CytoSMART Lux2 can be set up in minutes, enabling untrained users to quickly perform their own time-lapse recordings.
Images and videos can be easily accessed and retrieved from the CytoSMART cloud portal. Advanced functions, such as reporting of cell confluency, cell migration analysis and the option to use automatic confluency email alerts, can be applied to inform the user when certain culture conditions are reached (for example, once the cell culture has reached the desired confluency). Hence, the CytoSMART Lux2 can be used in many different ways to facilitate cell culture work and research.
In the following appnote several examples of applications of the CytoSMART Lux2 will be shown.
- Culturing cells in hypoxic conditions
- Standardizing cell culturing conditions
- The effect of confluency on transfection efficiency
Cell Motility Video Monitoring - Lux2
Movement of cells plays a critical role in the development of cancer. Analyzing the motility of cells in vitro is therefor important for many cancer researchers.
Live-cell imaging, and in particular label-free live-cell imaging, is well suited to capture dynamic processes in cell culture without potential side-effects of markers or dyes on the cells. In the text below we will discuss the suitability of the CytoSMARTTM
Lux for several assays relevant for cancer research
We will discuss
- Introduction to the Lux cell monitoring system
- Tube formation assays
- 2D migration assays
- 3D invasion assays