P1‐183: FLOW CYTOMETRIC CALCIUM FLUX MEASUREMENT IN ACUTELY‐ISOLATED BRAIN MYELOID CELLS

Abstract

Neuroinflammation in Alzheimer's disease (AD) is mediated primarily by microglia and brain-infiltrating monocytes/macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs)[1-3]. Effector functions of CNS-MPs are dependent of transient and sustained elevations in intracytosolic calcium (calcium flux)[4, 5]. Understanding the differences in the regulators of calcium flux in subsets of CNS-MPs as well as under homeostatic and neurodegeneration-associated activated states can help identify novel therapeutic strategies for immunomodulation in AD. In this study, we apply a flow-cytometric, ratiometric calcium flux assay to acutely-isolated CNS-MPs to contrast basal and stimulus-evoked calcium flux responses in resident microglia (CD45low) and CNS-macrophages (CD45high) CNS-MPs and in wild-type and AD mouse models (5xFAD). CNS-MPs from wild-type and 5xFAD mice were acutely isolated from adult mice and incubated with CD11b and CD45 flow-cytometric antibodies and Indo-1(30mins). Kinetic flow cytometric assay was performed (over 8 mins) to measure free intracellular calcium at baseline and following a series of immune stimuli: lipopolysaccharide(10uM), interferon gamma(100nM), ATP(10uM), thapsigargin(10uM) and ionomycin(1uM). Dependence of stimulus-evoked responses on Kir2.1 potassium channels were examined using a selective blocker ML133(100uM). FlowJo was used for kinetic and quantitative analyses of data. Acutely-isolated CNS-MPs demonstrated calcium flux in response to immune stimuli and thapsigargin, similar to BV2 microglia, establishing the validity of our flow assay.CD45high macrophages demonstrated higher basal calcium levels compared to CD45low microglia. Microglia showed higher calcium flux in response to all three immune stimuli compared to macrophages. Interferon-gamma response was seen only in microglia (p=0.02) Blockade of Kir2.1 channels significantly increased basal calcium levels in all CD45high macrophages, more evidently from 5xFAD mice (p=0.017). Additionally, there is higher thapsigargin-induced calcium flux (p=0.019) in CD45low microglia from 5xFAD mice as compared to wild-type microglia.