I recently discovered that BFA is used in flow cytometry, enabling the observation of intracellular cytokines that would be otherwise released. Is the same principle applicable to ROS? I would like to see them in neutrophils.
Thank you
Hello Stefano Gianoli
BFA is a reversible inhibitor of protein transport. Following treatment with BFA, the Golgi complex disassembles and redistributes into the endoplasmic reticulum within minutes. BFA is a potent, rapid, and reversible inhibitor of secretion. It inhibits the GTPase exchange factor acting on the ARF protein. ARF activates ADP-ribosylation factors to the Golgi complex.
BFA is mostly used in studies of membrane trafficking. It increases intracellular cytokine staining signals and is commonly used for intracellular staining of cytokines for flow cytometry. It blocks transport processes during cell activation and causes an accumulation of cytokines at the Golgi complex/ endoplasmic reticulum.
Besides its use in intracellular staining of cytokines, BFA shows antibiotic actions and induces apoptosis and autophagy in mammalian cells. The use of BFA to detect ROS is not known.
Nevertheless, if you are interested in measuring ROS production, the most widely used method is the use of the fluorescent probe 2’,7’ dichlorodihydrofluorescein diacetate (DCFH2-DA). This molecule is colorless and lipophilic. Diffusion of DCFH2-DA across the cell membrane allows it to be acted upon by intracellular esterases, which deacetylates it into DCFH2, rendering it cell impermeable. The actions of multiple types of ROS (hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radicals) on DCFH2 oxidize it into DCF which is fluorescent (reported Ex/Em: 485–500 nm/515–530 nm) and can be detected using a flow cytometer.
However, superoxide does not strongly react with DCFH2 but can react with another probe dihydroethidium (DHE) to yield the fluorescent product 2-hydroxyethidium as well as other fluorescent superoxide-independent oxidation products. The fluorescent products of DHE oxidation can be detected using an excitation wavelength of 518 nm and an emission wavelength of 605 nm. Include controls in the assay to have valid experimental results and conclusions.
You may want to refer to the articles attached below for detection of intracellular ROS by flow cytometry.
https://www.hindawi.com/journals/mi/2012/849136/
Article Flow Cytometric Measurement Of ROS Production In Macrophages...
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