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Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy
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JoVE 杂志 免疫与感染
Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

DOI:
10.3791/62733-v

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07:31 min

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August 19, 2021

•

,
1Department of Biochemistry & Microbiology, Faculty of Science,University of Victoria, 2Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Science,University of Calgary, 3Calvin, Joan and Phoebe Snyder Institute for Chronic Diseases,University of Calgary

Chapters

  • 00:05Introduction
  • 00:28Measuring Macropinosome pH
  • 02:06In Situ Calibration of Macropinosome pH
  • 03:04Measuring Oxidative Events within Macropinosomes
  • 04:22Measuring Protein Digestion within Macropinosomes
  • 05:30Results: Measuring the pH, Oxidative Events, and Protein Degradation in Macropinosomes using Dual-Fluorophore Ratiometric Microscopy
  • 06:59Conclusion

Summary

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We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

Tags

MacropinosomesPHRedoxDegradationDual-fluorophoreRatiometric MicroscopyFluoresceinTMRHBSSPotassium Rich SolutionNigericinRAW264.7 Cells

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