Characterization of microglia

Neuroimmune Function

Neuroimmunology is the study of nervous and immune system interactions, during development, homeostasis and in response to injury or infection. Immune cells can play a pivotal role in the modulation of neuronal networks, for example, synaptic pruning by microglia is a key component of neural plasticity. Injury, infection or loss of homeostasis can lead to neuroinflammation and activation of microglia, which in turn phagocytose dead or dying neurons and infective agents. Chronic neuroinflammation is believed to underlie a number of neurodegenerative disorders, such as Alzheimer’s disease, where prolonged activation of microglia is detrimental to neuronal networks.

The Incucyte® Live-Cell Analysis System enables extensive morphological and functional characterization of microglia. Fully automated image acquisition and analysis facilitates both the visualization and kinetic analysis of microglial efferocytosis of apoptotic neurons or phagocytosis of bioparticles. Microglial migration in response to chemotactic cytokines can be visualized and quantified with live-cell chemotactic migration and invasion assays. Non-perturbing reagents preserve cell morphology without the disruption and restrictions imposed by end-point analysis, thus allowing more data collection from every cell with uninterrupted incubation directly from inside your incubator at 96- and 384-well throughput.  

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Introducing Incucyte Assays for Neuroimmune Function

Key Advantages

Visualize and verify engulfment by microglia

• Observe microglial phagocytosis with images and movies using pH sensitive fluorescent probes.
• Approach is amenable to engulfment of apoptotic cells, cell debris, protein aggregates and bioparticles.

Figure 1. Visualize and verify engulfment by microglia. Time-lapse visualization of iPSC-derived microglia (Axol BioSciences) engulfing pHrodo Orange labeled apoptotic Neuro2A cells.  Images verify the entry of an apoptotic target cell into the cytoplasm of the microglia.  Neuro2A target cells were labeled with the Incucyte pHrodo Orange Cell Labeling Kit and apoptosis induced with staurosporine.

Real-time quantification of phagocytosis and efferocytosis by microglia

• Automated segmentation of fluorescent signal elicited from the Incucyte® pHrodo® Orange Reagent
• Real-time analysis of phagocytosis response to either apoptotic Neuro2A cells or β amyloid aggregates conjugated with the Incucyte pHrodo Orange Cell Labeling Kit
• Evaluate mechanism of action for phagocytosis modulators
  
  

Figure 2. Real-time quantification of phagocytosis by microglia. Representative fluorescent images of iPSC-derived microglia (Axol Bioscience) engulfment of pHrodo labeled apoptotic neurons (Neuro-2A). An increase in intracellular orange fluorescence was observed as target cells were internalized into acidic phagosomes; the segmentation mask is shown as the cyan outline. Efferocytosis of pHrodo labeled Neuro2A cells is cell number-dependent. Phagocytosis of pHrodo labeled Aβ aggregates was time- and concentration-dependent.

Figure 3. Evaluation of the mechanism of phagocytosis modulation. BV-2 effector cells efferocytose apoptotic Neuro2A cells (left hand panels) or E. coli bioparticles (middle panels).  Cytochalasin D (top panels) elicits a concentration-dependent inhibition of both efferocytosis and phagocytosis, yielding IC50 values of 0.16 µM and 1.5 µM, respectively.  In contrast, cilengitide, an inhibitor of aVb3 and aVb5 integrins, selectively attenuates efferocytosis (IC50 value 0.17 µM), while inducing little or no effect on phagocytosis at the highest concentration tested (100 µM). These data support the role of integrins in the cell interactions required for efferocytosis, but not in the phagocytosis of bacteria.

Evaluate microglial chemotaxis and migration

• Observe microglial chemotaxis with images and movies
• Utilize only 1000 to 5000 cells per 96-well chemotaxis plate, ideal for low cell usage of rare, expensive and primary cell populations

Figure 4. Evaluate microglial chemotaxis and migration. Image of iPSC-derived microglia (CDI) in an Incucyte ClearView Plate. Pores are circled in blue (left). Microglia demonstrate a time- and concentration-dependent migration towards Complement component 5a (C5a).

Monitor morphological changes following differentiation or activation

• Track changes in morphology during the differentiation of monocytes to microglia

Figure 5. Visualization of the morphological changes of iPSC-derived monocytes (Axol BioScience) differentiated to microglia. Note the varied appearance of microglia.  After 12 days red arrows highlight cells with a ramified appearance, whereas the yellow arrows show the larger flat amoeboid cells. Cells were fed with differentiation media every 2 days for 14 days, and Phase images acquired at 20x magnification every 6 hours.

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