FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins
Abstract Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engul...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-09-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-41448-7 |
| _version_ | 1797558019896115200 |
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| author | Lasse Dissing-Olesen Alec J. Walker Qian Feng Helena J. Barr Alicia C. Walker Lili Xie Daniel K. Wilton Indrani Das Larry I. Benowitz Beth Stevens |
| author_facet | Lasse Dissing-Olesen Alec J. Walker Qian Feng Helena J. Barr Alicia C. Walker Lili Xie Daniel K. Wilton Indrani Das Larry I. Benowitz Beth Stevens |
| author_sort | Lasse Dissing-Olesen |
| collection | DOAJ |
| description | Abstract Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease. |
| first_indexed | 2024-03-10T17:25:28Z |
| format | Article |
| id | doaj.art-8e693c8aa0b84be6a2269dca6bec6129 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-10T17:25:28Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-8e693c8aa0b84be6a2269dca6bec61292023-11-20T10:12:16ZengNature PortfolioNature Communications2041-17232023-09-0114111510.1038/s41467-023-41448-7FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteinsLasse Dissing-Olesen0Alec J. Walker1Qian Feng2Helena J. Barr3Alicia C. Walker4Lili Xie5Daniel K. Wilton6Indrani Das7Larry I. Benowitz8Beth Stevens9Department of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurosurgery, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurosurgery, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurosurgery, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterDepartment of Neurology, Boston Children’s Hospital, F.M. Kirby Neurobiology CenterAbstract Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease.https://doi.org/10.1038/s41467-023-41448-7 |
| spellingShingle | Lasse Dissing-Olesen Alec J. Walker Qian Feng Helena J. Barr Alicia C. Walker Lili Xie Daniel K. Wilton Indrani Das Larry I. Benowitz Beth Stevens FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins Nature Communications |
| title | FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| title_full | FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| title_fullStr | FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| title_full_unstemmed | FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| title_short | FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| title_sort | feast a flow cytometry based toolkit for interrogating microglial engulfment of synaptic and myelin proteins |
| url | https://doi.org/10.1038/s41467-023-41448-7 |
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