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 stroke2,3,4. CNS-MPs, along with astrocytes, pericytes and ependymal cells, have phagocytic functions5,6. 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 connections7,8,9,10. 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 roles11,12,13,14. 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 AD4,9. 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 diseases15,16, 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 microscopy18,19,20. It is well-established that CNS-MPs, when maintained in long cultures, can dramatically change their morphology and transcriptional profiles21,22. 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 approaches9,23. 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
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The study was supported by NIH awards to Dr. Rangaraju (NINDS K08 NS099474-1 and R01 NS114130-01A1) and Dr. Rayaprolu (NIA F32AG064862). This study was supported in part by the Emory Flow Cytometry Core (EFCC), one of the Emory Integrated Core Facilities (EICF) and is subsidized by the Emory University School of Medicine. Additional support was provided by the Georgia Clinical & Translational Science Alliance of the NIH (UL1TR002378). The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH.
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. |
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