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Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages
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Imunologia e Infecção
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JoVE Journal Imunologia e Infecção
Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

DOI:
10.3791/63162-v

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08:41 min

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April 06, 2022

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1Department of Pediatrics, Division of Hematology-Oncology, Department of Molecular and Cellular Biology, Texas Children’s Hospital William T. Shearer Center for Human Immunobiology,Baylor College of Medicine

Capítulos

  • 00:04Introduction
  • 00:51Preparation of Cells for Electroporation
  • 02:11Nucleofection of Caspase BiFC Components into Human Monocyte-Derived Macrophages
  • 04:41Treatment of Transfected MDM and Caspase BiFC Data Acquisition
  • 06:37Results: Differentiation of CD14-Monocytes and Transfection of Human Monocyte-Derived Macrophages
  • 08:12Conclusion

Summary

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This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Tags

Keywords: Inflammatory CaspasesProximityHuman Monocyte-derived MacrophagesVisualizationMolecular-levelPrimary Immune CellsOligomerizationMicroscopic MethodologiesTrypsin-EDTAPBSCell NumberHemocytometerReporter PlasmidCaspase BiFC PlasmidsNucleofectionElectroporation

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