CLP-d2

Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of ³²P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.

Collagen-induced arthritis (CIA) is an autoimmune murine model of rheumatoid arthritis in which immunization with type II collagen (CII) emulsified in an adjuvant induces a T cell- and autoantibody-driven inflammatory arthritis characterized by synovial hyperplasia, immune cell infiltration, and joint destruction. The model typically relies on genetically susceptible mouse strains (e. g. , DBA/1) and reproduces key features of human rheumatoid arthritis, including anti-collagen immune responses and progressive joint inflammation. Disease onset generally occurs within ~3-4 weeks after immunization, depending on antigen/adjuvant combinations and protocol variation. The immunopathology is driven by adaptive immune activation against CII, leading to systemic and local joint inflammation mediated by pro-inflammatory cytokines and effector immune cells, making CIA a standard preclinical platform for evaluating immunomodulatory and anti-arthritic interventions.

Lipopolysaccharide (LPS)-induced endotoxemia is a widely used in vivo model of acute systemic inflammation in which LPS, a Gram-negative bacterial endotoxin, activates innate immune signaling primarily through TLR4, leading to rapid and transient induction of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in circulation and tissues. This cytokine surge is commonly used as a measurable readout of systemic inflammatory activation and immune dysregulation, and is typically assessed within hours after intraperitoneal LPS administration in mouse models of endotoxemia. The model captures key features of systemic inflammatory response syndrome, including cytokine release, immune cell activation, and downstream tissue responses, and has been used to evaluate anti-inflammatory interventions such as cytokine modulation, lipid mediators, and immune cell-targeting therapies.

Cotton pellet granuloma is a classical in vivo chronic inflammation model used to evaluate the anti-inflammatory potential of test substances by measuring their ability to inhibit granuloma tissue formation around an implanted foreign body (cotton pellet) in rodents. The method is based on the biological response to a sterile implanted material, which induces proliferative phase inflammation characterized by fibroblast proliferation and collagen-rich granuloma formation, and the final readout reflects the extent of chronic inflammatory tissue growth surrounding the pellet. In multiple preclinical pharmacological evaluations, inhibition of cotton pellet-induced granuloma formation has been used as an indicator of anti-inflammatory activity in both synthetic and natural product screening contexts.

Carrageenan-induced paw edema is an acute inflammation model in which intraplantar injection of carrageenan induces localized inflammatory swelling characterized by vascular permeability, leukocyte infiltration, and production of inflammatory mediators such as prostaglandins and cytokines, making it widely used to evaluate anti-inflammatory agents in vivo. The resulting paw volume or thickness increase is quantified over time as a direct readout of inflammatory intensity and drug efficacy, typically reflecting cyclooxygenase-mediated prostaglandin-driven edema formation and immune cell recruitment in peripheral tissue[20].

The NLRP3 inflammasome is a cytosolic innate immune signaling platform that integrates priming signals and danger-signal activation to promote caspase-1 activation, maturation of IL-1β and IL-18, and gasdermin D-mediated pyroptotic cell death. The core experimental logic is to determine whether inflammatory disease phenotypes are driven by increased NLRP3 expression, ASC-containing inflammasome assembly, caspase-1 cleavage, GSDMD cleavage, and extracellular release of IL-1β/IL-18 rather than by nonspecific cell injury alone. The pathway is strongly linked to inflammation-related disease phenotypes because monosodium urate crystals activate NALP3/NLRP3 inflammasome signaling in gout-like crystal inflammation, cholesterol crystals activate NLRP3 inflammasomes in atherogenesis models, and DSS-induced intestinal inflammation has been reported to involve NLRP3 inflammasome activity. However, experimental colitis studies also show context-dependent protective effects of NLRP3 inflammasome co

Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.

Osteoclast differentiation is an in vitro induction assay in which monocyte/macrophage-lineage precursors are exposed to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), generating multinucleated osteoclasts that are commonly identified by tartrate-resistant acid phosphatase (TRAP) staining and functionally confirmed by resorption pits on dentin, bone, or mineralized substrates. M-CSF supports survival and expansion of osteoclast precursors, while RANKL binding to RANK drives osteoclast commitment, fusion, maturation, and resorptive function; osteoprotegerin inhibits this pathway by binding RANKL and preventing RANK activation. The main readouts are the number of TRAP-positive multinucleated cells, formation of F-actin rings, and resorbed surface area; TRAP-positive multinucleated cells indicate osteoclast differentiation, whereas pit formation on dentin, bone, or mineralized coating indicates functional bone-resorbing activity.

Pyroptosis is a lytic inflammatory cell-death pathway executed by gasdermin pores, most classically through inflammasome-mediated activation of caspase-1, cleavage of gasdermin D, membrane pore formation, LDH release, and secretion of IL-1β and IL-18. The canonical pathway is commonly modeled by priming cells with an inflammatory signal such as LPS to induce pro-IL-1β and inflammasome components, followed by an activation signal such as ATP or nigericin to activate NLRP3, ASC speck formation, caspase-1 cleavage, GSDMD cleavage, cytokine release, and pyroptotic membrane rupture. The non-canonical pathway is triggered when cytosolic LPS activates mouse caspase-11 or human caspase-4/5, leading to GSDMD cleavage and pyroptosis, and this can secondarily activate NLRP3-dependent IL-1β release. Pyroptosis is linked to inflammatory injury, infection, cancer, liver disease, ocular disease, placental inflammation, and other disease phenotypes, but unresolved questions include which gasdermin fam