Incucyte Applications

Glia Characterization



Glial cells, such as astrocytes, microglia, and oligodendrocytes, play a number of important roles in the central nervous system (CNS), such as maintaining homeostasis, actively involved in synaptogenesis, or protecting and supporting neurons. Recently, the need to understand the role of glia in health and disease have become pivotal, and novel research tools and approaches are been developed from an increased glia-centric perspective. It is now recognized that glia cultures are an important tool for basic and translational research, with the use of primary cultures or iPSCs being considered more relevant for investigating human diseases. However, models are largely limited to end-point morphological and immunohistochemical methods. Glia research will benefit from the development of technological tools and translational in-vitro models to increase insight into glia cell growth, morphology, function, and ultimately their roles in CNS diseases.

Key Advantages

Key Advantages

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Quantify glia growth, morphology and pharmacology

Use kinetic, high-throughput measurements to monitor dynamic biological changes.

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Multiplex to gain deeper insights

Monitor cell cycle progression, modulation and cell viability using non-perturbing reagents.

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Visualize and quantify glia movement

Obtain real-time cell movement measurements with fully automated analysis.

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Utilize physiologically relevant glia models

Study glia supportive functions with relevant cell models.

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Quantify glia growth, morphology and pharmacology

Use kinetic, high-throughput measurements to monitor dynamic biological in different brain regions

Glia Characterization Figure 1

Figure 1. Temporal monitoring of brain region astroglia revealed differences in cell growth and morphology.  Cortex, Hippocampus and Cerebellum astroglia were seeded in 96-well plates at 2,000 cells/well. Proliferation and morphology was monitored over 10 days. Images show cultures at 30-40% confluence (2 days, cortical or 4 days, hippocampal and cerebellar), where ramified morphological phenotype is quantified (pink and browns masks). Time-course profile compares growth amongst brain regions and reveals cortical astrocytes have the fastest rate of growth. Glia ramification is compared overtime with cerebellum astrocytes yielding the highest ramification by 96h (68.3 ± 1.8) followed by hippocampal (50.6 ± 1.5) and cortical (12.3 ± 0.7). Maximum ramification for each well is also shown (variability plot). Data presented as Mean ± SEM (24 replicates) and images were captured at 10x magnification.

Pharmacological effect of ionomycin on cerebellar astrocytes

Glia Characterization Figure 2

Figure 2. Ionomycin-induced calcium increase effects cell growth and health in cerebellar astrocytes. Cerebellum astrocytes were seeded in 96-well plates at 10,000 cells/well and treated with calcium ionophore ionomycin (0.01 – 10 µM) in media supplemented with cell health reagent Incucyte® Annexin V NIR (Sartorius; 0.5%). Proliferation and cell health were monitored over 10 days. Time-course shows the kinetic effect of ionomycin on cell growth. Concentration-response curves compare the effect of ionomycin on cell growth and health, with a concentration-dependent decrease in confluence and increase in apoptosis observed suggestive of toxic effects of ionomycin. Representative images show effects on growth (phase) and cell health (NIR) at 24 h. Data presented as Mean ± SEM (3 replicates) and images were captured at 10x magnification.

Multiplex to gain deeper insights

Monitor cell cycle progression, modulation and cell viability using non-perturbing reagents to untangle complex modulation

Glia Characterization Figure 3

Figure 3. Okadaic Acid (OKA) induced cell cycle arrest can be distinguished from apoptosis in astrocytic cell line. T98G astrocytic cells expressing Incucyte® Cell Cycle Green|Orange lentivirus were seeded at 4,000 cells/well in a 96-well plate. Cells were treated with protein phosphatase inhibitor OKA (50 – 0.14 nM) in the presence of cell health reagent Incucyte® Annexin V NIR (0.5%; Sartorius) to enable the effects on both cell cycle arrest and cell viability to be determined. Images were acquired at 10X using the Incucyte® Cell-by-Cell analysis module to allow cells to be individually segmented, with representative phase and/or fluorescence (green, orange, NIR) phase videos shown for vehicle and OKA over 2 days post-treatment. Time-courses, showing the populations of cells in S|G2|M (green) or G1 (orange) and the population of Annexin V positive cells (teal) for vehicle or OKA, revealed OKA (5.56 nM) caused an arrest in S|G2|M within 16 h and this remained stable until approximately 36 h when the number of apoptotic cells began to increase. Concentration-response curves indicate rapid efficacy of OKA at 20 h on populations in different phases and a delayed effect on cell viability (Annexin V Positive) at 48h. Data presented as Mean ± SEM, 6 replicates.

Visualize and quantify glia movement

Obtain real-time cell movement measurements with fully automated analysis to evaluate post-injury in real-time

Glia Characterization Figure 4

Figure 4. Post-injury migration and pharmacological inhibition of astrocytes from different brain regions.  Primary (Cortex, Hippocampus, Cerebellum) or iPSC (CDI, Fujifilm) astrocytes were seeded in Incucyte® ImagelockT96-well plates at 30,000 cells/well and precise, reproducible wounds were created with the Incucyte® Woundmaker. Images were acquired using the Incucyte® live-cell analysis System. Phase videos show migration of hippocampal cultures following injury over 3 days, with segmentation shown for the initial scratch wound (blue mask) and wound during closure (light yellow mask), and allow for morphological assessment of cells. Time-course profiles compare rate of wound closure for different brain regions with cerebellar astroglia migrating the fastest. For pharmacological studies, cortical astroglia were incubated with ionomycin (10 – 0.01 µM) prior to wounding and concentration-dependent inhibition of migration is observed (IC50 = 1.67 µM). Data presented as Mean ± SEM, 24 replicates.

Quantify glia chemotaxis in real-time with fully automated analysis

Glia Characterization Figure 5

Figure 5. T98G-WT astrocyte chemotactic migration towards FBS. T98G-WT cells were plated in the top chamber of the Incuycte® ClearView 96-Well Chemotaxis plate coated with PDL (0.1 mg/mL) at a density of 1,000 cells/well. Once the cells had adhered, fetal bovine serum (FBS; 10 - 0.12%) was added to the bottom chamber as a chemoattractant. Images, and respective masks, are representative of the top and bottom side of the membrane at 48 h post-addition. Time-course and bar chart at 48h indicate a concentration-dependent increase in chemotactic migration through the pore with increasing levels of FBS (EC50 = 3%). Data presented as Mean ± SEM, 10 replicates.

Utilize physiologically relevant glia models

Study glia supportive functions with relevant cell models, iPSC-derived or primary cells, in mono- or co-culture to evaluate astrocytic supportive functions

Glia Characterization Figure 6

Figure 6. iPSC astrocytes in neuronal co-culture enhance neurite development and stabilises network activity. Human iPSC-derived neurons were seeded as a mono- or co-culture with astrocytes (25K for neurons, 10K cells/well for astrocytes; Talisman Therapeutics). Neurons were infected with Incucyte® Neurolight or Neuroburst Orange (Sartorius) to assess neurite development or monitor spontaneous neuronal activity over time, respectively. Time-courses reveal co-cultures developed greater neurite branching and outgrowth compared to mono-cultures (NL: 147 ± 2.5 vs. 72 ± 2.5 mm/mm2 , respectively at 228h). Traces represent calcium fluctuation of all active objects within the field of view. Bar graphs show quantification of active objects, correlation (connectivity), burst rate and duration at 23 days. Co-cultures yield increased active objects and display a reduced frequency of longer-lasting calcium events compared to mono-cultures, indicating network stabilisation, whilst connectivity is unaffected. Results suggest supportive functions of iPSC astrocytes within a co-culture, where neurons display enhanced morphological phenotype and develop more functionally mature networks. Mean ± SEM, 24 replicates.

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