Protocol for rapid obtention and fractionation of anaerobic bacterial conditioned media to study calcium signaling in enteroendocrine cells

• Published in: STAR Protocols
• Main Authors: Amorim Neto, Dionísio Pedro, de Castro Fonseca, Matheus
• Format: Article in Journal/Newspaper
• Language: unknown
• Published: Cell Press 2022
• Subjects: Cell Biology; Microbiology; Microscopy; General Immunology and Microbiology; General Biochemistry; Genetics and Molecular Biology; General Neuroscience; sami
• Online Access: https://doi.org/10.1016/j.xpro.2022.101486; https://www.ncbi.nlm.nih.gov/pmc/PMC9214868

Gut microbiota influences neurodevelopment, behavior and contributes to neurodegenerative disorders. One possible mechanism is the direct modulation of calcium (Ca²âº) signaling and protein homeostasis in enteroendocrine cells (EECs), a component of the gut epithelium. Here, we present a protocol to isolate fractions of conditioned media (CM) from the anaerobic bacteria Akkermansia muciniphila and the utilization of this CM to monitor Ca²⺠fluctuation in EECs by imaging. This protocol can be adapted and applied to various bacterial cultures and cell types. For complete details on the use and execution of this protocol, please refer to Amorim Neto et al. (2022). © 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Available online 18 June 2022, Version of Record 18 June 2022. We thank Dr. Silvana Rocco, Dr. Maurício Sforça and Dr. Rodrigo Alborghetti (LNBio, Brazil) for helping with NMR data collection, analysis, and interpretation. We also thank Dr. Sami Yokoo for her scientific contribution. The authors would like to acknowledge support of INFABIC, Unicamp and the Brazilian Biorenewables National Laboratory (LNBR – CNPEM) for providing access to its facilities. We also acknowledge the Mass Spectrometry Laboratory and Nuclear Magnetic Resonance Laboratory at Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Brazil, for their support with the mass spectrometry and NMR analysis. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) under the grant numbers 2018/20014-0 and 2019/24511-0. Author contributions. Conceptualization, M.C.F. and D.P.A.N.; Formal Analysis, D.P.A.N. and M.C.F.; Investigation, D.P.A.N.; Resources, M.C.F.; Writing, D.P.A.N. and M.C.F.; Funding Acquisition, M.C.F. The authors declare no competing interests. Data and code availability. This study did not generate datasets. Published - ...