Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Summary

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Abstract

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent Aβ fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Introduction

The innate immune cells of the central nervous system (CNS) are predominantly comprised of microglia and infiltrating monocytes/macrophages, together called as the CNS-mononuclear phagocytes (CNS-MPs)¹. CNS-MPs are implicated in neurodegenerative diseases such as Alzheimer’s disease (AD), neuroinflammatory disorders, and stroke^2,3,4. CNS-MPs, along with astrocytes, pericytes and ependymal cells, have phagocytic functions^5,6. In their homeostatic state, CNS-MPs are involved in constant surveillance of the local microenvironment, along with phagocytic clearance of apoptotic cell debris and proteins, and synaptic pruning to remodel neuronal connections^7,8,9,10. In neurodegenerative conditions such as AD, CNS-MPs adopt distinct disease-associated molecular and functional phenotypes which can play pathological roles including clearance of aggregated amyloid-beta (Aβ) and neuronal elements, as well as inflammatory cytokine and factor release, resulting in complex pro-inflammatory, detrimental as well as anti-inflammatory, protective roles^11,12,13,14. Phagocytosis of macroparticles, cellular debris, proteins and other infectious particles by CNS-MPs are mediated by distinct receptors expressed on their surface⁹. Disruption in these phagocytic pathways can lead to defective clearance and progressive accumulation of Aβ ultimately leading to progressive neuronal damage in AD^4,9. While advances in molecular profiling of CNS-MPs using transcriptomic and proteomic approaches have provided invaluable insights into the molecular heterogeneity within CNS-MPs in neurological diseases^15,16, functional characterization of the phagocytic properties of CNS-MPs and their subsets is currently lacking. Functional characterization of the phagocytic properties of CNS-MPs can complement molecular profiling strategies, facilitate better functional phenotyping, and assist in assessing the efficacy of therapeutics that can rectify defective phagocytosis in disease models¹⁷.

Traditional phagocytosis assays for CNS-MPs include incubating primary microglia along with fluorescent substrates such as Aβ or latex/polystyrene particles. Phagocytosis is then studied as a function of uptake of the fluorescent substrate using immunofluorescence microscopy^18,19,20. It is well-established that CNS-MPs, when maintained in long cultures, can dramatically change their morphology and transcriptional profiles^21,22. This hinders studying the phagocytic properties of CNS-MPs in their representative state in the CNS. A functional flow cytometry assay to profile acutely-isolated live CNS-MPs can provide a rapid assessment of phagocytic properties and can sample a much larger pool of CNS-MPs than microscopy approaches^9,23. Furthermore, flow cytometry eliminates the need for maintaining the CNS-MPs in culture as well as provides a platform to study phagocytotic properties in different sub-populations of CNS-MPs. This manuscript describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. Adapting phagocytosis assays using flow cytometry allows rapid multiplexing of the phagocytic phenotype of CNS-MPs coupled with immune phenotyping, thereby providing insights into heterogeneity of phagocytic properties within CNS-MPs at the single cell resolution.

Protocol

All mouse studies were conducted after obtaining approval from the Emory University’s Institutional Animal Care and Use Committee (IACUC), and in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. 1. Preparation on the day of isolation Prepare an ice bucket to keep all reagents, buffers, and cell suspensions cold throughout the isolation procedure. Place the bottle of 1× phosphate-buffered saline (P…

Representative Results

To exemplify typical results of phagocytic uptake of microspheres and Aβ42 fibrils by acutely-isolated CNS-MPs, acutely-isolated CNS-MPs were obtained from the ipsilateral hemisphere following transient middle cerebral arterial occlusion (MCAO)8. For details regarding phagocytic properties of CNS-MPs in the MCAO model, please refer to prior publications8. Briefly, after a 72 h recovery, CNS-MPs were acutely isolated from fresh brain using mechanical dissociation follow…

Discussion

Flow-cytometry is a technique that can detect the expression of proteins or markers of interest on the cell surface or in intracellular compartments using fluorescently labeled probes (typically antibodies) to label these markers of interest. Cell-surface or intracellular antibodies are typically conjugated with laser-excitable fluorochromes that have unique emission spectra. Cells incubated with these antibodies can be categorized into multiple sub-populations based on patterns of expression of these markers. CNS-MPs co…

Materials

10x Hank's balanced salt solution(10x HBSS)	ThermoFisher	14065056	Store at 4 °C. Used to make SIP as described in the protocol
1x Hank's balanced salt solution(1x HBSS)	ThermoFisher	14175095	Store at 4 °C. Used to make 35% SIP as described in the protocol
1x Phosphate Buffered Saline (PBS)			https://www.sigmaaldrich.com/technical-documents/protocols/biology/western-blotting/buffers-recipes/10x-phosphate-buffered-saline.html Store 10xPBS at Room temperature. Prepare fresh 1xPBS by diluting one part 10xPBS to nine parts of MilliQ dH20 and store it at 4 °C for an hour before use. Make sure the pH of the 1xPBS is 7.0 before refridgeration.
35% SIP			To make 20 mL of 35% SIP, use 7 mL of SIP and 13 mL of ice-cold 1× HBSS. Keep on ice until further processing
APC-Cy7 rat anti-CD11b	BD Pharmingen	557657	Store at 4 °C or keep on ice when in use. Sheild from light. Flow cytometry dilution – 1:100
FITC rat anti mouse CD145	BD Pharmingen	553080	Store at 4 °C or keep on ice when in use. Sheild from light. Flow cytometry dilution – 1:100
LIVE/DEAD Fixable Blue Dead Cell Stain Kit	ThermoFisher	L34961	Store at -20 °C. Add 50ul of DMSO to one tube of the dye, vortex throughly and centrifuge at maximun speed for 1 min. Make 10µL aliquots and store them at -20 °C. Use a fresh aliquot for each experiment and add to the samples at 1:500 dilution.
OneComp eBeads compensation beads	ThermoFisher	01-1111-42	Store at 4 °C, throughly vortex the tube before adding to the sample tube(s), keep on ice when in use.
PE-Cy7 rat anti mouse CD145	BD Pharmingen	552848	Store at 4 °C or keep on ice when in use. Sheild from light. Flow cytometry dilution – 1:100
Percoll pH8.5-9.5	Sigma	P1644	Store at 4 °C. To make 10 mL of Standard Isotonic Percoll (SIP): Use 9 mL of cold Percoll and 1 mL of cold 10× HBSS.
Phycoerythrin-conjugated microspheres	ThermoFisher	F13083	Store at 4 °C or keep on ice when in use.
β-Amyloid (1 – 42), HiLyte Fluor 488 – labeled, Human	AnaSpec	AS-60479-01	The final concentration of Ab fibrils is 200µM which is then diluted prior to use. Store at -20 °C. Prepare fresh working solution for each experiment.