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Journal of Neuroinflammation

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Open Access 01-12-2024 | Research

Impact of microglia isolation and culture methodology on transcriptional profile and function

Authors: Mark Mizrachi, Betty Diamond

Published in: Journal of Neuroinflammation | Issue 1/2024

Abstract

Background

Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate five different microglia isolation methods—three variants of microglia isolation from neonatal mice and two variants of microglia isolation from adult mice—on transcriptional profile and response to HMGB1.

Methods

Microglia from neonatal mice, age 0–3 days (P0–P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6–8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al. in Front Cell Neurosci 17:1082180, 2023), or acute isolation using CD11b beads (Bordt et al. in STAR Protoc 1:100035, 2020. https://doi.org/10.1016/j.xpro.2020.100035). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ concentration in supernatant by ELISA and assessing gene expression patterns using bulk mRNA sequencing.

Results

All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription and secretion of TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from neonates than from adult mice, with sparse changes among the three MGC culturing conditions. Additionally, cultured microglia derived from adult and microglia derived from MGCs from neonates display transcriptional signatures corresponding to an earlier developmental stage.

Conclusion

These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

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Title: Impact of microglia isolation and culture methodology on transcriptional profile and function
Authors: Mark Mizrachi
Betty Diamond
Publication date: 01-12-2024
Publisher: BioMed Central
Published in: Journal of Neuroinflammation / Issue 1/2024
Electronic ISSN: 1742-2094
DOI: https://doi.org/10.1186/s12974-024-03076-w

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Impact of microglia isolation and culture methodology on transcriptional profile and function

Abstract

Background

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

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