Impact of microglia isolation and culture methodology on transcriptional profile and function
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 f...
| Asıl Yazarlar: | , |
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| Materyal Türü: | Makale |
| Dil: | English |
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BMC
2024-04-01
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| Seri Bilgileri: | Journal of Neuroinflammation |
| Online Erişim: | https://doi.org/10.1186/s12974-024-03076-w |
| _version_ | 1827284152825675776 |
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| author | Mark Mizrachi Betty Diamond |
| author_facet | Mark Mizrachi Betty Diamond |
| author_sort | Mark Mizrachi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T09:51:03Z |
| format | Article |
| id | doaj.art-662fc770c1f149d2a7e0f1fb5d5dd6a9 |
| institution | Directory Open Access Journal |
| issn | 1742-2094 |
| language | English |
| last_indexed | 2024-04-24T09:51:03Z |
| publishDate | 2024-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Neuroinflammation |
| spelling | doaj.art-662fc770c1f149d2a7e0f1fb5d5dd6a92024-04-14T11:23:28ZengBMCJournal of Neuroinflammation1742-20942024-04-0121111010.1186/s12974-024-03076-wImpact of microglia isolation and culture methodology on transcriptional profile and functionMark Mizrachi0Betty Diamond1Feinstein Institutes of Molecular Medicine, Feinstein Institutes for Medical ResearchFeinstein Institutes of Molecular Medicine, Feinstein Institutes for Medical ResearchAbstract 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.https://doi.org/10.1186/s12974-024-03076-w |
| spellingShingle | Mark Mizrachi Betty Diamond Impact of microglia isolation and culture methodology on transcriptional profile and function Journal of Neuroinflammation |
| title | Impact of microglia isolation and culture methodology on transcriptional profile and function |
| title_full | Impact of microglia isolation and culture methodology on transcriptional profile and function |
| title_fullStr | Impact of microglia isolation and culture methodology on transcriptional profile and function |
| title_full_unstemmed | Impact of microglia isolation and culture methodology on transcriptional profile and function |
| title_short | Impact of microglia isolation and culture methodology on transcriptional profile and function |
| title_sort | impact of microglia isolation and culture methodology on transcriptional profile and function |
| url | https://doi.org/10.1186/s12974-024-03076-w |
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