Responses during cell preparation for functional analyses in mouse bone marrow-derived cultured mast cells
Masaru Murakami1, Teruo Ikeda, Yoshii Nishino
1Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan.
Insights
Mast cell preparation methods can transiently induce gene expression of plasminogen activator inhibitor-1 (PAI-1) and AP-1 components. Researchers must consider these procedural effects when interpreting in vitro mast cell data.
Area of Science:
- Immunology
- Cell Biology
Background:
- Murine bone marrow-derived cultured mast cells (BMMCs) are crucial for in vitro mast cell function studies.
- Standard cell preparation techniques may inadvertently influence experimental outcomes.
Purpose of the Study:
- To investigate the impact of cell preparation procedures on BMMC gene expression.
- To identify specific molecular pathways affected by cell collection and density adjustment.
Main Methods:
- BMMCs were subjected to standard preparation protocols including centrifugation and density adjustment.
- Gene expression levels of plasminogen activator inhibitor-1 (PAI-1) and AP-1 components (c-fos, c-jun, junB) were analyzed.
- Protein dephosphorylation of MAP kinases (ERK, p38, JNK) was assessed.
- Effects of okadaic acid, a protein phosphatase inhibitor, were examined.
Main Results:
- Cell preparation transiently induced PAI-1 and AP-1 component gene expression.
- PAI-1 transcript levels correlated with cell density.
- Okadaic acid pretreatment enhanced PAI-1 gene expression.
- Preparation induced MAP kinase dephosphorylation via protein phosphatase 1 (PP1) and 2A (PP2A) activation.
Conclusions:
- Standard BMMC preparation can activate specific signaling pathways, affecting gene expression.
- Careful consideration of cell preparation artifacts is essential for accurate interpretation of in vitro mast cell research.
- Findings highlight the need for optimized protocols to minimize experimental variability.
Abstract:
Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e., cell collection by centrifugation and the subsequent adjustment and culture of cell density at the desired concentrations, transiently induced gene expression of plasminogen activator inhibitor-1 (PAI-1) and the AP-1 components (c-fos, c-jun, and junB). The level of PAI-1 gene transcript was closely related to the cell density and the gene expression was enhanced by pretreatment with okadaic acid, an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A). The cell preparation procedure also caused dephosphorylation of MAP kinases, i.e., ERK, p38, and JNK, resulting from PP1/PP2A activation. In view of the cell responses to the cell preparation procedure itself, care is needed in the interpretation of in vitro data using BMMCs.
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