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Journal article
Microenvironmental Conditions and Serum Availability Alter Primary Human Macrophage NF-κB Inflammatory Response and Function
Journal of leukocyte biology, v 117(7), qiaf071
Jul 2025
PMID: 40401596
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Macrophages are central to innate immunity and are routinely used in vitro to examine molecular mechanisms contributing to innate immune signaling. However, there is a lack of consensus within the field for optimal in vitro culturing methods, and it is not well understood whether differences in culture conditions produce incongruent outcomes. Here, we compared the effects of commonly used culture medium compositions on TLR4-mediated pro-inflammatory activity in primary human monocyte-derived macrophages (hMDM) isolated from healthy blood donors. hMDM were cultured in fetal bovine serum (FBS)-containing or FBS-free conditions in either DMEM, RPMI, or in Macrophage-Serum Free Medium (M-SFM). LPS-mediated immune response was measured through NF-κB activation and cytokine and chemokine secretion, which were muted in M-SFM cultures compared to DMEM and RPMI cultures. FBS supplementation increased total cytokine secretion in response to LPS but also showed higher baseline secretion, suggesting a pro-inflammatory phenotype. Moreover, M-SFM cultures exhibited less phagocytosis compared to DMEM and RPMI cultures. Morphologic analysis of unstimulated hMDM revealed the highest cell area and length-to-width ratio in M-SFM compared to DMEM or RPMI cultures. FBS-free and M-SFM conditions produced distinct transcriptional profiles compared to media supplemented with FBS, most notably in cell cycle pathways and lipid homeostasis, respectively. Overall, DMEM and RPMI produce comparable morphologic and functional results, albeit with some small differences, while M-SFM produces a muted inflammatory response in macrophages. These data demonstrate that in vitro microenvironment drives differential inflammatory outcomes in human macrophages and is a critical component of experimental design in this cell type.
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Details
- Title
- Microenvironmental Conditions and Serum Availability Alter Primary Human Macrophage NF-κB Inflammatory Response and Function
- Creators
- Breana Channer - Drexel UniversityMarzieh Daniali - Drexel UniversityLexi Sheldon - University of Nebraska Medical CenterKaty Emanuel - University of Nebraska Medical CenterYash Agarwal - Drexel UniversityTaylor Kist - Drexel UniversityBrian J Murphy - Drexel UniversityMeng Niu - University of Nebraska Medical CenterWill Dampier - Drexel UniversityHoward Fox - University of Nebraska Medical CenterPeter J Gaskill (Corresponding Author) - Drexel University
- Publication Details
- Journal of leukocyte biology, v 117(7), qiaf071
- Publisher
- Oxford University Press
- Number of pages
- 16
- Grant note
This work was supported by grants from the National Institutes of Drug Abuse, the National Institutes of Mental Health and the National Institutes of Allergy and Infectious Disease, DA057337 and DA058051 (P.J.G.), T32-MH079785 (supporting B.C.), F30AI179472 (B.C.), U01 DA053624 (H.F.), P30-MH092177 (supporting W.D.), as well as the Department of Pharmacology and Physiology at Drexel University College of Medicine.
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Microbiology and Immunology; Pharmacology and Physiology
- Web of Science ID
- WOS:001524496200001
- Scopus ID
- 2-s2.0-105010317744
- Other Identifier
- 991022053803504721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Cell Biology
- Hematology
- Immunology