Aug 04, 2023
Bone Marrow Derived Macrophage (BMDM) differentiation and maintenance
- Narayana Yadavalli1,2,3,4,5,
- Shawn M. Ferguson1,2,3,4,6,5
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
- 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. Bone Marrow Derived Macrophage (BMDM) differentiation and maintenance. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw7bwl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82373
Keywords: Macrophages, L929 media, Bone marrow, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes the isolation of bone marrow derived macrophages.
Attachments
724-1815.docx
17KB
Materials
Reagents required
- L929 conditioned media
- Penicillin/StreptomycinThermo Fisher ScientificCatalog #Invitrogen 15140-122
- Gibco™ Fetal Bovine SerumFisher ScientificCatalog #16-140-071
- Gibco™ DMEM/F-12 HEPESThermo Fisher ScientificCatalog #11330032
- GlutaMAX™ SupplementGibco - Thermo FisherCatalog #35050061
Note
Note: For mouse bone marrow-derived macrophage (BMDMs) primary cultures, each experiment involved age and sex matched C57BL/6 mice between 3-9 months of age. Lrrk2KO (Lrrk2tm1.1Mjff) and G2019S (Lrrk2tm1.1Hlme) homozygous knockin mice were obtained from The Jackson Laboratory76.
Euthanize mice via CO2 or isoflurane inhalation and cervical dislocation.
Collect femurs and flush cavities with 5 mL ice-cold PBS.
Then, collect bone marrow cells by centrifugation 0.3 rcf, 00:05:00 .
5m
Resuspend the resulting pellet and differentiate for 6 days in culture media containing: DMEMF12 supplemented with 20% FBS, 20% L929 conditioned media, 1% penicillin-streptomycin and 1% GlutaMAXTM.