Characterization of 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.

• 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.

• 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).

• 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.

• 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.