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Measurement of Reactive Oxygen Species by Fluorescent Probes in Pancreatic Cancer Cells

  • Yongde LuoEmail author
  • Dan Wang
  • James L. Abbruzzese
  • Weiqin LuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1882)

Abstract

Pancreatic cancer is a highly lethal disease and is projected to become the second leading cause of cancer-related death by 2020. Among the different subtypes, pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. The genetic landscape of PDAC shows nearly ubiquitous mutations of KRAS. However, expression of KRAS somatic mutants alone is insufficient to drive PDAC. Redox deregulation may contribute significantly to KRAS-mediated PDAC. Thus, measurement of cellular reactive oxygen species (ROS) levels is essential to determine how oxidative stress affects mutant KRAS and modulates intracellular signaling pathways leading to the change of cellular functions and the development of PDAC. Here we describe the protocol for comparative measurement of several key forms of ROS, including intracellular and mitochondrial levels of superoxide as well as extracellular H2O2 and general cellular ROS, with oxidation-sensitive fluorescent probes using flow cytometry in pancreatic cancer cells or mutant KRAS transformed cells.

Key words

Pancreatic cancer Mutant KRAS Reactive oxygen species (ROS) NADPH oxidase Superoxide Hydrogen peroxide Redox Mitochondria Flow cytometry Fluorescent probe 

Notes

Acknowledgment

This work is supported by the Start-up Funds from Stony Brook University [W. L], the Pilot Project Grant from the Department of Medicine at Stony Brook University [W. L], a P20 grant from NIH [5P20CA192994-02 to W.L as a coinvestigator], and the National Science Foundation of China [81370957 to Y.L].

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center for Cancer and Metabolism Research, Institute for Life ScienceWenzhou UniversityZhejiangChina
  2. 2.School of Pharmaceutical ScienceWenzhou Medical UniversityZhejiangChina
  3. 3.Proteomics and Nanotechnology Laboratory, Center for Cancer and Stem Cell Biology, Institute of Biosciences and TechnologyTexas A&M University Health Science CenterHoustonUSA
  4. 4.Center BioTherapeutics Ltd. Co.HoustonUSA
  5. 5.Division of Gastroenterology and Hepatology, Department of MedicineStony Brook University School of MedicineStony BrookUSA
  6. 6.Division of Medical Oncology, Department of Medicine, Duke Cancer InstituteDuke UniversityDurhamUSA

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