Characterization of Microglia
Neuroimmunology is the study of nervous and immune system interactions, during development, homeostasis and in response to injury or infection. 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. 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 microglia efferocytosis of apoptotic neurons or phagocytosis of bioparticles, and real-time quantification of migration or invasion in response to chemotactic cytokines. The Incucyte® Kinase Akt Assay enables visualization and quantification of dynamic changes in Akt activity in neuroinflammatory models. 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.
Introducing Incucyte® Assays for Neuroinflammation
Incucyte Phagocytosis Assay
- Generate quantitative, reproducible and specific measurements of engulfed cells
- Visualize and validate dynamic phagocytic cell clearance of cellular debris and dead neurons with images and movies
- Perform non-perturbing labeling of targets with the Incucyte® pHrodo® Orange Cell Labeling Kit
- Efficiently study the full time course of phagocytosis using the model of your choice in microplate format
Incucyte Chemotaxis Assay
- Link real-time chemotaxis measurements to visual assessments of morphology and gain deep phenotypic insight
- Analyze sensitive and rare cells with a cell sparing, highly reproducible 96-well assay
- Measure label-free or labeled cell migration without fixing, staining or cell scraping steps
- Turnkey solution includes purpose-built software and novel, optically clear Incucyte ClearView Plates
Incucyte® Kinase Akt Assay
- Express a stable, genetically-encoded kinase activity biosensor using Incucyte® Kinase Akt Green/Red Lentivirus
- Bridge the gap between single, target-focused biochemical assays and lower-throughput cell-based experiments for the identification of novel treatments
- Gain new insights with non-perturbing sensitive detection of dynamic changes in Akt activity in live cells over time
- Quantify Akt kinase activity and proliferation within the same assay in your neuroinflammatory model of choice
- Visualize and verify engulfment by microglia with the Incucyte® Phagocytosis Assay
- Real-time quantification of phagocytosis and efferocytosis by microglia
- Evaluate microglial chemotaxis
- Monitor morphological changes following differentiation or activation
- Visualize and quantify dynamic changes in Akt activity
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 pHrodo® Orange Cell Labeling Kit for Incucyte® and apoptosis induced with staurosporine.
Real-time quantification of phagocytosis and efferocytosis by microglia
- Automated segmentation of fluorescent signal elicited from the pHrodo® Orange Cell Labeling Dye for Incucyte®
- Real-time analysis of phagocytosis response to either apoptotic Neuro2A cells or β amyloid aggregates conjugated with the pHrodo® Orange Cell Labeling Kit for Incucyte®
- 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 Incucyte® Clearview 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.
Visualize and quantify dynamic changes in Akt activity
- Assess real-time changes in Akt activity in neuroinflammatory microglial models using non-perturbing Incucyte® Kinase Akt Green/Red Lentivirus.
Figure 6. Direct and indirect inhibitory compound effects on microglial Akt activity. HMC3 microglia cells were labeled with the Incucyte® Kinase Green/Red Lentivirus, a genetically-encoded fluorescent kinase translocation reporter whose subcellular localization is phosphorylation dependent. Cells seeded into a 96-well plate were treated with inhibitors targeting Akt (MK2206), and both upstream PI3K (LY294002 and Wortmannin) and downstream mTOR (PP242). HD Phase contrast and fluorescent images were acquired using the Incucyte® Live-Cell Analysis System and the nuclear translocation ratio (NTR), an indicator of Akt activity, was quantified using integrated software.
The plate view displays the NTR response over 24 hours where all four compounds display concentration-dependent inhibition of Akt activity, as indicated by a decrease in NTR. PI3K inhibitors LY294002 and Wortmannin showed rapid inhibition followed by a recovery to basal Akt activity levels, whilst allosteric Akt Inhibitor MK2206 and mTOR inhibitor PP242 attenuates Akt activity in a sustained manner. Transformation of the data into concentration-response curves for NTR at 6 hours post-treatment indicates that MK2206 has the most potent effect. Images of the green fluorescence channel show that the fluorescence in untreated (vehicle) cells is distributed across both the cytoplasm and the nucleus, however the MK2206 treated cells show distinct localization in the nucleus, indicative of Akt inhibition.
|pHrodo® Orange Cell Labeling Kit for Incucyte® Phagocytosis Assays|
|pHrodo® Red Cell Labeling Kit for Incucyte® Phagocytosis Assays|
Incucyte® Chemotaxis Software Module
Incucyte® Clearview 96-well Chemotaxis Plate
Incucyte® Clearview 96-well Chemotaxis Plate – Case of 10 Plates
Incucyte® Kinase Akt Green/Red Lentivirus
Incucyte® SX5 Live-Cell Analysis System