Kumatakenin

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.

Annexin V-based apoptosis assays rely on the detection of phosphatidylserine (PS) externalization from the inner leaflet of the plasma membrane to the outer leaflet, an early biochemical hallmark of apoptosis. Fluorescently labeled Annexin V binds PS in a calcium-dependent manner, enabling identification of early apoptotic cells by flow cytometry or fluorescence microscopy. When combined with a membrane-impermeant DNA-binding dye (e. g. , propidium iodide), this approach allows discrimination between viable (Annexin V−/dye−), early apoptotic (Annexin V+/dye−), and late apoptotic or necrotic (Annexin V+/dye+) cell populations by assessing membrane integrity and PS exposure.

Cell migration is a method that plays an important role in wound healing, cell differentiation, embryonic development, etc.

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

The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.

Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics.

TUNEL staining detects DNA strand breaks by using terminal deoxynucleotidyl transferase to add labeled nucleotides to exposed 3′-OH DNA termini, generating either microscopic staining in fixed cells or tissue sections, or fluorescence/cytometric signal in cell suspensions. TUNEL positivity reflects DNA fragmentation but should not be interpreted alone as definitive apoptosis, because TUNEL can also label necrotic, autolytic, mechanically damaged, or DNA-repair-associated DNA breaks.

Dextran sulfate sodium (DSS)-induced colitis is generated by administering DSS in mouse drinking water, producing epithelial injury, barrier disruption, weight loss, diarrhea, fecal blood, colon shortening, histologic mucosal damage, and inflammatory mediator changes; the model is mainly used to study acute or chronic intestinal inflammation resembling selected features of ulcerative colitis. DSS injury is interpreted through clinical and tissue readouts rather than a single molecular endpoint: daily body weight, stool consistency, and bleeding are combined into a disease activity index, while colon length, histology, cytokines, myeloperoxidase activity, intestinal permeability, and tight-junction markers provide complementary measures of inflammation and barrier damage.

TNBS-induced colitis is produced by intrarectal delivery of 2,4,6-trinitrobenzene sulfonic acid in ethanol, where ethanol disrupts the mucosal barrier and TNBS haptenates colonic proteins, generating immune-mediated colonic inflammation with weight loss, diarrhea, ulceration, transmural injury, inflammatory-cell infiltration, and cytokine responses. The model is used as an experimental intestinal inflammation model with Crohn’s disease–like features, especially when Th1-type responses, IL-12–dependent inflammation, chronic relapsing inflammation, or fibrosis-related endpoints are studied.

Autophagy is a process in which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy related gene (Atg). Microtubule-associated proteins light chain 3 (LC3) is recognized as autophagy marker, which transfers from cytoplasmic LC3 (LC3-I) to membrane type (LC3-II). LC3-II/I ratio could be detected by Western Blot and fluorescence microscopy.

Apoptosis is a regulated, generally non-lytic cell-death pathway that removes unwanted, damaged, infected, or abnormal cells through coordinated morphological changes, caspase activation, DNA fragmentation, and membrane remodeling. The intrinsic apoptosis pathway is controlled mainly by mitochondrial outer membrane permeabilization, BCL-2 family proteins, cytochrome c release, apoptosome formation, caspase-9 activation, and downstream executioner caspase-3/7 activation. The extrinsic apoptosis pathway is initiated by death receptors such as Fas, TNFR, and TRAIL receptors, which recruit adaptor proteins and activate caspase-8 before engaging executioner caspases or mitochondrial amplification through BID cleavage. Apoptosis is linked to many phenotypes, including cancer cell killing, tissue homeostasis, immune regulation, neurodegeneration, infection response, and treatment-induced cytotoxicity; unresolved questions include how apoptosis interacts with necroptosis, pyroptosis, ferroptos

Primary human monocytes can be differentiated ex vivo into monocyte-derived macrophages by culturing purified blood monocytes for approximately 5-7 days in macrophage-supporting cytokine conditions; M-CSF commonly yields CD14^high/CD163^high macrophages, while GM-CSF yields a phenotypically distinct macrophage population, so the cytokine condition should be chosen according to the downstream model. The readout of successful differentiation is a combined change in morphology, adherence, surface phenotype, and function: differentiated macrophages become adherent, enlarge, acquire macrophage-associated markers such as CD14, CD68, CD163, CD206, or HLA-DR depending on culture condition, and show increased phagocytic capacity compared with starting monocytes.

TEER measures electrical resistance across epithelial or endothelial monolayers cultured on permeable supports, and the readout reflects ionic conductance through the cell barrier, especially the paracellular pathway regulated by junctional integrity. TEER can be measured without destroying the monolayer and is commonly used before or during transport, permeability, barrier-disruption, and barrier-maturation experiments. TEER values are influenced by biological maturation and technical conditions; reported factors include temperature, medium formulation, passage number, electrode geometry, membrane properties, and junctional length during early monolayer maturation. Therefore, TEER should be interpreted with blank-insert subtraction, area normalization, repeated readings, and, when possible, orthogonal barrier readouts such as FITC-dextran flux or tight-junction staining.

Cell invasion is the ability of cells to migrate from one area to another via the extracellular matrix. Cell invasion is the response of normal and cancer cells to chemical and mechanical stimuli. Before migrating to a new region, the extracellular matrix is degraded by proteases within the cell. Cell invasion often occurs during wound repair, vascularization and inflammation, abnormal tissue invasion, and tumor cell metastasis.

Lysosome and acidic-vesicle live-cell staining detects acidic intracellular compartments by using membrane-permeant acidotropic probes that accumulate in low-pH vesicles, including lysosomes, late endosomes, autolysosomes, and acidic phagosomes. LysoTracker staining is commonly used as an intensity-based readout of acidic lysosomal compartment abundance or enlargement, while acridine orange produces green fluorescence in less concentrated compartments and red fluorescence after concentration-dependent accumulation in acidic vesicular organelles. Loss or reduction of acridine-orange red signal can be used as a readout of lysosomal membrane permeabilization or reduced acidic-vesicle integrity. This protocol is designed for live cultured cells and can be adapted for fluorescence microscopy, high-content imaging, plate-reader readout, or flow cytometry when the selected literature supports the readout. Because these dyes report acidotropic accumulation rather than lysosome identity alone,

Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death characterized by lethal lipid peroxidation and sensitivity to suppression by iron chelators or lipophilic radical-trapping antioxidants. The core pathway links cystine uptake through system Xc−, glutathione availability, GPX4-dependent detoxification of phospholipid hydroperoxides, iron-dependent oxidative reactions, and polyunsaturated-phospholipid metabolism into a cell-death program that is biochemically and morphologically distinct from apoptosis, necrosis, and autophagy. The ferroptosis pathway is experimentally linked to phenotype through chemical and genetic perturbation. Erastin induces ferroptosis by inhibiting cystine uptake through system Xc− and weakening antioxidant defenses, while GPX4 inhibition or depletion causes lipid peroxide accumulation and ferroptotic cancer-cell death. ACSL4 and oxidizable arachidonoyl- or adrenoyl-containing phosphatidylethanolamines shape ferroptosis sensitivity by con

Macroautophagy is a conserved lysosome-dependent degradation pathway in which cytoplasmic material is sequestered into double-membrane autophagosomes and delivered to lysosomes for degradation and recycling. The pathway supports cellular homeostasis during nutrient limitation, organelle stress, protein-aggregate accumulation, infection, differentiation, and tissue remodeling by coupling cargo sequestration, autophagosome maturation, lysosomal fusion, and degradation of cargo-derived macromolecules. The core molecular sequence includes initiation by nutrient- and stress-regulated autophagy machinery, autophagosome nucleation, LC3/ATG8-family conjugation to autophagosomal membranes, cargo selection through receptors such as SQSTM1/p62, autophagosome-lysosome fusion, and lysosomal degradation. LC3 was identified as a mammalian homolog of yeast Atg8 that localizes to autophagosomal membranes after processing, and p62/SQSTM1 was shown to connect ubiquitinated cargo with autophagic degradati