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Morphological, immunological and metabolic features of different activation states in BV-2 microglial cell line

Introduction: Microglia cells are resident macrophages in the brain and represent the first line of defense against endogenous and exogenous stimuli. They respond to stimuli by activation, which encompasses both immune and metabolic changes in the cell. Two broad activation phenotypes are classically activated M1 phenotype and the alternatively activated M2 ramified phenotype, which is present in physiological conditions. Limitations of our current in-vitro research is the permanent activated state of the cells. The aim of our research is establishing a model of ramified BV-2 microglial cells for the purpose of having similar conditions to those found physiologically in-vivo. Methods: Research was performed on the immortalized BV-2 microglial cell line. Cells were cultivated in 10%, 5% and 0% fetal calf serum (FCS) DMEM, supplemented with L-glutamine and penicillin/streptomycin. We have used several methods to assess the activation of microglial cells. Light and immunofluorescent microscopy was used to study cell morphology, with the latter also used for studying the expression of cell phenotype markers (CD206, Arginase-1, CD16/32, iNOS, hypoxia inducible factor-1). Flow cytometry was used to assess the expression cell surface markers CD86 and CD16/32. Furthermore, we assessed the cell metabolic profile using Seahorse (Agilent technologies). Results: Cultivating cells in varied FCS percentage had a significant impact in cell phenotype, both in morphology and function. Cells cultivated without FCS displayed a ramified phenotype, while the cells cultivated in 10% FCS displayed an activated phenotype. Metabolism measurements revealed a quiescent phenotype for cells cultivated without FCS, while cells cultivated in 10% FCS had an energetic (aerobic and anaerobic) profile. Both cell cultures had the same response to stressors from baseline. Conclusion: BV-2 microglia cultivated without FCS is morphologically and functionally similar to ramified microglia in- vivo, while retaining the same capability to respond to stressors compared to the cells cultivated with FCS.

Microglia, cell cultivation, immunometabolomics

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