A Simple Microplate Assay for Reactive Oxygen Species Generation and Rapid Cellular Protein Normalization

Abstract

2',7'-dichlorofluorescein (DCF) and derivatives are commonly used as fluorescent indicators of a broad spectrum of reactive oxygen species (ROS) generation in cell-based assays. However, there are numerous challenges inherent to the utilization of DCF probes for intracellular microscopic analysis, including photostability and probe efflux. Plate spectroscopy is comparatively simple and scalable compared to microscopy or flow cytometry-based acquisition, however is often subject to artefacts, including those introduced by thermal gradients and normalization methods. In this protocol we demonstrate a simple and sensitive plate spectrometry-based protocol utilizing the probes H2DCFDA and sulforhodamine B. The rapid sulforhodamine B assay (SRB) for cellular protein allows for a stable endpoint measurement of total cell population while also preserving morphology, can be combined or run in parallel with any other assay for normalization of readout to cell mass, and complemented by microscopic scoring of cell number and nuclear count. The oxidative stress and normalisation methods may enhance fields of research investigating cell differentiation, stress, or toxicity..

Anti-Inflammatory and Anti-Acne Effects of Hamamelis virginiana Bark in Human Keratinocytes

“…490 nm/em 535 nm. The results were normalized for the protein content determined by the sulforhodamine B (Merck Life Science, Milano, Italy) assay, according to Ng and Ooi [ 29 ].…”

Uptake and Immunomodulatory Properties of Betanin, Vulgaxanthin I and Indicaxanthin towards Caco-2 Intestinal Cells

“…The generation of reactive oxygen species (ROS) within Caco-2 cells was determined in 96-well plates with the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH 2 -DA), which is intracellularly deacetylated and oxidised to highly fluorescent 2′,7′-dichlorofluorescein (DCF) [ 32 , 33 ]. DCFH 2 -DA (30 µM) dissolved in PBS with 1% FBS ( v / v ) was applied to the confluent Caco-2 cells and incubated for 40 min at 37 °C.…”

“…After washing, cells were treated with BET, VUL and IND (5–80 μM) for 1 h and subsequently stressed with H 2 O 2 (200 μM) for a further 1 h. The plate was scanned using Tecan Spark 10M TM with excitation λ of 485 nm and emission λ of 535 nm. Subsequently, the protein content of each well was determined by SRB assay [ 33 ]. Briefly, the medium was removed and the cells were washed with PBS and submerged with 50 μL of 0.004% SRB solution ( w / v ) in 10% trichloroacetic acid ( w / v ).…”

Green Synthesis of Gelatin‐Lipid Nanocarriers Incorporating Berberis aristata Extract for Cancer Therapy; Physical Characterization, Pharmacological and Molecular Modeling Analysis

“…[47] The estimation depends on the electrostatic binding of SRB dye, a negatively charged fluorescent dye, to basic amino acid residues of a protein under acidic conditions. [48] The appearance of bright color gives the indication of binding of SRB dye to intracellular proteins. It depends on the quantity of protein content in a cell so representing a concentration dependent principle.…”