Adult Murine Microglia Purification and Stimulation
Microglia are the resident immune-effector cells in the central nervous system and amount to 10-15 % of all brain cells. Microglial activation typically occurs in response to brain injury, neurodegeneration, and infection, resulting in altered function and the release of inflammatory mediators which may be beneficial or detrimental, depending on the microenvironmental context.
Microglia are highly sensitive to changes in their environment. This is particularly evident during isolation and culturing of microglia in vitro with serum. Although numerous methods of isolating intact microglia are widely used, the majority lead to cells that are both highly proliferative and amoeboid, hallmarks of microglial activation. However, more physiological culture methods are now available that better recapitulate the in vivo environment.
Purification of adult murine microglia:
Adult mice brains are dissected and dissociated into single cells. Microglia are subsequently sorted by magnetic selection and purity confirmed by CD11b/P2RY12 expression using flow cytometry.
Microglia isolated from adult mice are highly pure, express microglia phenotypic markers, and adopt a ramified morphology in defined culture conditions. Cultured microglia release pro-inflammatory cytokines upon stimulation with LPS that can be inhibited using corticosteroids (e.g. prednisolone, PRD). These assays can be used to both probe ex vivo microglial activation status from healthy or diseased mice and assess whether immunomodulating therapeutics can dampen or enhance microglial activation in vitro.
In vitro stimulation of adult murine microglia:
Purified microglia are cultured for 6 days in conditioned culture medium supplemented with defined astrocytic factors. Cultured microglia exhibit a ramified small bodied morphology turning amoeboid during microglial activation, following LPS stimulation. Images below were taken using the IncuCyte® S3 platform.
Microglial Activation: Phagocytosis Assays
Microglia phagocytosis occurs under homeostasis and is often perturbed in disease states. Growing evidence suggests that retarded clearance of misfolded protein or aberrant synaptic pruning can exacerbate disease pathology, for example in Alzheimer’s Disease.
Adult murine microglia cultured under serum-free conditions efficiently phagocytose bio-particles and their phagocytic function can be either enhanced or suppressed depending on the mechanistic intervention. In vitro screening utilizing adult microglia derived from healthy or disease model tissue can provide information on mode of action and impact on cell function of therapeutics targeting microglial cells.
Phagocytosis assay methodology:
Microglia are purified from adult murine brains and pre-incubated with the therapeutic. Target materials (e.g., beads or bio-particles) are labeled with the pH sensitive dye pH-Rodo-Red and applied to cultured microglia. Phagocytosis is kinetically monitored over 9 hours using the IncuCyte S3 platform.
Phagocytosis, in response to distinct molecular signals, is a key function of microglia required to maintain homeostasis and is a key readout of their normal function. Therapeutic manipulation of this function can drive the clearance of aberrant disease material such as dysfunctional cells or aggregated proteins such as α-Synuclein and β-amyloid. Similarly, in different disease circumstances, reducing overactive phagocytic clearance may be therapeutically beneficial.
Frequently Asked Questions (FAQs) for Murine Microglial Activation Assays
What are the benefits of using adult microglia over more commonly used neonatal cells?
Current microglial models of neuroinflammation include microglial cell lines or primary microglia prepared from embryonic or neonatal mice. Increasingly, studies report diverse microglial phenotypes that are linked to development showing distinct molecular signatures. Commonly studied microglia cell lines and embryonic or neonatal-derived microglia show differing responses to stimuli and express strikingly different molecular signatures compared to adult microglia. The use of adult microglia is especially pertinent in the field of neurodegenerative diseases where aging is a critical parameter.
How can IncuCyte® Phagocytosis Assays help me to test my therapeutic?
Measuring phagocytosis in real-time using the IncuCyte system allows you to visualize and quantify microglial phagocytosis activity over time, adding a higher level of mechanistic insight which can complement endpoint phagocytosis readouts by methods such as flow cytometry or immunofluorescence. A plethora of target materials such as synthetic beads, protein aggregates, or cells can be labelled with high specificity allowing a highly-tailored assay approach. This method can be readily scaled up to test several compounds in response to different stimuli in comparison to a positive or negative control drug. Together, this assay offers a flexible, bespoke platform to test microglial function which can be tailored to your therapeutic and target of interest.