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A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

ABSTRACT: Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring low concentrations of ROS in nonphagocytic cells. We found that dissolving luminol in NaOH, rather than DMSO, increased the H₂O₂-induced CL signal and that the addition of 4-iodophenylboronic acid (4IPBA) further increased CL intensity. Our new system also increased the rate and intensity of the CL signal in phorbol 12-myristate 13-acetate- (PMA-) treated HT-29 colon cancer cells compared to those in luminol only. We were able to quantify ROS levels from both cells and media in parallel using an H₂O₂ standard. A significant benefit to our system is that we can easily measure stimulus-induced ROS formation in a real-time manner and also investigate intracellular signaling pathways from a single sample simultaneously. We found that PMA induced tyrosine phosphorylation of protein tyrosine kinases (PTKs), such as focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), and Src, and increased actin stress fiber formation in a ROS-dependent manner. Interestingly, treatment with either N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) reduced the PMA-stimulated phosphorylation of these PTKs, implicating a potential role in cellular ROS signaling. Thus, our newly optimized CL assay using 4IPBA and a chemiluminescent imaging method provides a simple, real-time, and low-cost method for the quantification of low levels of ROS.

SUBMITTER: Kim JS

PROVIDER: S-EPMC6594271 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

Kim Jun-Sub JS Jeong Kyuho K Murphy James M JM Rodriguez Yelitza A R YAR Lim Ssang-Taek Steve SS

Oxidative medicine and cellular longevity 20190611

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring ...[more]

PMID: 31285782

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Project description:SignificanceStudying cerebral hemodynamics may provide diagnostic information on neurological conditions. Wide-field imaging techniques, such as laser speckle imaging (LSI) and optical intrinsic signal imaging, are commonly used to study cerebral hemodynamics. However, they often do not account appropriately for the optical properties of the brain that can vary among subjects and even during a single measurement. Here, we describe the combination of LSI and spatial-frequency domain imaging (SFDI) into a wide-field quantitative hemodynamic imaging (QHI) system that can correct the effects of optical properties on LSI measurements to achieve a quantitative measurement of cerebral blood flow (CBF).AimWe describe the design, fabrication, and testing of QHI.ApproachThe QHI hardware combines LSI and SFDI with spatial and temporal synchronization. We characterized system sensitivity, accuracy, and precision with tissue-mimicking phantoms. With SFDI optical property measurements, we describe a method derived from dynamic light scattering to obtain absolute CBF values from LSI and SFDI measurements. We illustrate the potential benefits of absolute CBF measurements in resting-state and dynamic experiments.ResultsQHI achieved a 50-Hz raw acquisition frame rate with a 10×10 mm field of view and flow sensitivity up to ∼4 mm/s. The extracted SFDI optical properties agreed well with a commercial system (R2≥0.98). The system showed high stability with low coefficients of variations over multiple sessions within the same day (<1%) and over multiple days (<4%). When optical properties were considered, the in-vivo hypercapnia gas challenge showed a slight difference in CBF (-1.5% to 0.5% difference). The in-vivo resting-state experiment showed a change in CBF ranking for nine out of 13 animals when the correction method was applied to LSI CBF measurements.ConclusionsWe developed a wide-field QHI system to account for the confounding effects of optical properties on CBF LSI measurements using the information obtained from SFDI.

Dataset Information

A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

Publications

A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

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