Guidelines for anti-inflammatory assays in RAW264.7 cells

Abstract

In recent years, research on various bioactive compounds with anti-inflammation effects has become increasingly active. Evaluating anti-inflammatory activity is well established in cell models represented by RAW264.7 cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and cell counting kit-8 (CCK-8) assays are commonly used to detect the effects of drugs or deleterious substances on cytotoxicity, cell viability, and survival. RAW264.7 cells release nitric oxide (NO) when exposed to inflammatory stimuli, so their anti-inflammatory capacity can be assessed by detecting the NO content in cell culture supernatants, and the Griess method is one of the commonly used methods for determining NO concentration. RAW264.7 cells release a variety of inflammation-associated cytokines, such as the TNF-α and IL-1β, with ELISA being the standard method to detect the release of these inflammatory factors. For mRNA expression levels of genes associated with inflammation, real-time PCR (Quantitative Real-time PCR, qPCR) is commonly used. In addition, the well-known western blotting assay can be used to detect the expression levels of proteins associated with inflammatory signaling pathways. In this guideline, we discuss the basic principles and practices of the above experimental methods, which provide a valuable reference for the anti-inflammatory experimental methods in cellular models such as the RAW264.7 cells.