Raddeanin A

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.

Multiplex immunofluorescence IHC detects multiple protein biomarkers in one tissue section by sequential antibody staining, HRP-mediated tyramide fluorophore deposition, heat-mediated antibody stripping, nuclear counterstaining, multispectral imaging, spectral unmixing, and digital cell phenotyping; TSA deposits fluorophore near the antigen so the fluorescence signal remains after primary and secondary antibodies are removed, enabling repeated staining cycles, including with antibodies from the same host species. Classic FFPE tumor immune-profiling applications use panels such as CD3, CD8, CD68/CD163, FOXP3, PD-1, PD-L1, pancytokeratin, Ki67, and DAPI to identify tumor cells, immune-cell subsets, checkpoint-marker expression, co-expression phenotypes, cell density, and spatial relationships in the tumor microenvironment.

Human pluripotent stem cell endothelial differentiation is based on stepwise developmental patterning: early activation of WNT/GSK3β inhibition promotes mesodermal or vascular progenitor entry, followed by endothelial specification using VEGF-related signaling, BMP4, FGF2, Notch modulation, or cAMP depending on the published protocol. Endothelial differentiation is read out by acquisition of CD31, CD34, VE-cadherin/CD144, KDR/VEGFR2, vWF, Tie2, NOS3, acetylated LDL uptake, tube/network formation, barrier function, and in vivo vessel-forming capacity where tested.

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

Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive functions and loss of specific types of neurons and synapses. Alzheimer's symptoms can be simulated in mice by injecting drugs (such as Aβ) or genetically modified.

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.

Flow cytometric DNA-content cell-cycle staining measures the fluorescence intensity of DNA-bound fluorochromes in single cells or nuclei to estimate DNA content distributions, allowing assignment of populations to G0/G1, S, and G2/M phases by DNA histogram deconvolution. Propidium iodide (PI) intercalates into DNA, and PI fluorescence is proportional to cellular DNA content when staining is performed under conditions that make DNA accessible and minimize non-DNA signal. Cells with G2/M DNA content are expected to show approximately twice the fluorescence intensity of G0/G1 cells, while S-phase cells occupy intermediate fluorescence values. PI-based DNA-content analysis can also detect cells with fractional DNA content, often reported as sub-G1, when DNA fragmentation and extraction during staining reduce retained DNA signal in apoptotic cells. DAPI is an alternative DNA fluorochrome for univariate DNA-content analysis, while bivariate approaches combining DNA content with proliferation

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

Endothelial tube formation assay evaluates the ability of endothelial cells to attach, migrate, align, and organize into capillary-like networks when cultured on gelled basement membrane extract or Matrigel; the readout is the morphology and quantity of tube-like networks, which reflects an in vitro endothelial morphogenesis step related to angiogenesis. Basement membrane extract/Matrigel provides laminin-rich extracellular matrix cues that support endothelial differentiation into capillary-like structures, but it can contain biologically active growth factors, so growth-factor-reduced matrix is preferred when testing defined angiogenic stimulators or inhibitors.

The organotypic 3D invasion assay measures carcinoma-cell invasion into a fibroblast-remodeled extracellular matrix, usually collagen I with or without basement-membrane matrix, under an air-liquid or grid-supported culture condition; the readout is invasion depth, invaded area, or an invasion index from histological or fluorescence images. This assay models stromal regulation of invasion because fibroblasts or CAFs remodel matrix, generate tracks, and can lead collective carcinoma-cell invasion; the resulting cancer-cell penetration into the gel reflects tumor-stroma-ECM interactions rather than migration on a rigid 2D substrate.

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

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.

The Matrigel/ECM Transwell invasion assay measures the ability of cells to move toward a chemoattractant while crossing an extracellular-matrix barrier placed on a porous membrane; therefore, the readout reflects both chemotactic motility and matrix invasion rather than migration alone. Matrigel is a basement-membrane-rich matrix derived from Engelbreth-Holm-Swarm mouse sarcoma and has been used as a reconstituted basement membrane barrier in chemoinvasion assays. The assay readout is generated by quantifying cells that reach the underside of the insert membrane or lower compartment after incubation, commonly by staining and counting invaded cells or by fluorescence-based quantification.

Endothelial cell migration and angiogenic sprouting assays are in vitro (and partially ex vivo-adapted) functional models that quantify the ability of endothelial cells to undergo coordinated migration, extracellular matrix invasion, and multicellular organization into capillary-like sprouts in response to pro-angiogenic stimuli such as VEGF, bFGF, or conditioned microenvironments. These assays are used to model early angiogenic events including tip-cell formation, directional migration, and lumen-like sprout extension, which collectively reflect angiogenic activation and vascular morphogenesis processes observed in vivo.

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

The 3D collagen/hydrogel matrix invasion assay is based on embedding cells within or on top of a three-dimensional fibrillar extracellular matrix (typically type I collagen or collagen-rich hydrogels) to model cell migration through a physiologically relevant physical barrier. In this system, invasive behavior is quantified by measuring the ability of cells to degrade, remodel, and migrate through the 3D matrix architecture, which better reflects in vivo tissue invasion compared to 2D migration assays. Collagen-based 3D matrices provide structural cues such as fiber alignment and porosity that influence cell motility and integrin-mediated adhesion, enabling observation of collective or single-cell invasion modes depending on matrix density and organization.

The spheroid/tumor organoid invasion assay measures outward movement of cancer cells from a compact 3D aggregate into an extracellular matrix, usually collagen I, basement membrane matrix, or mixed collagen-Matrigel hydrogels; the readout is generated by bright-field, fluorescence, confocal, or time-lapse imaging of cell egress, invasion area, invasion distance, dispersion, protrusion formation, basement-membrane perforation, or cell trajectories. The assay reflects cell-cell cohesion, cell-matrix adhesion, matrix remodeling, protease-dependent invasion, contractility, and invasion behavior in a 3D microenvironment rather than migration on a flat 2D surface.

Ex vivo organotypic tissue slice cultures are based on maintaining thin, viable tissue sections at an air-liquid interface to preserve native cytoarchitecture and local cell-matrix interactions, enabling observation of cell behavior such as migration and tissue infiltration within a physiologically relevant 3D microenvironment. The method relies on maintaining tissue viability on porous membrane supports, allowing diffusion of nutrients and oxygen while preserving structural integrity for extended culture periods, which makes it suitable for studying dynamic cellular processes in intact tissue contexts such as cell movement and tissue remodeling. .

Patient-derived organoid (PDO) invasion assays are based on the ability of epithelial tumor organoids to self-organize in three-dimensional extracellular matrix (ECM) hydrogels (commonly Matrigel) and to recapitulate key aspects of in vivo tissue architecture, including polarity, proliferation, and invasive outgrowth when exposed to permissive microenvironmental cues. In this system, invasion is operationally defined as the emergence of multicellular protrusions, collective budding, or single-cell dissemination from the organoid core into the surrounding ECM, reflecting epithelial-mesenchymal plasticity and matrix remodeling capacity. Organoid morphology and invasive behavior are typically monitored using brightfield or confocal microscopy over time, enabling quantitative assessment of invasion area, protrusion number, and structural disruption of the organoid spheroid architecture.

Vascular/branching fractal analysis quantifies the geometric complexity of vessel trees or vascular networks from segmented 2D images, commonly by converting vessels into binary and/or skeletonized maps and estimating fractal dimension using box-counting or related approaches. Fractal dimension is interpreted as an image-derived readout of vascular branching complexity, space filling, or density, and has been applied to retinal photographs, fluorescein angiography, OCT angiography, capillary perfusion maps, and in vitro Matrigel angiogenesis networks. The assay readout is generated from vessel-positive pixels after image preprocessing, vessel segmentation, binarization, and optional skeletonization; reported outputs include fractal dimension, vessel density, branchpoint density, endpoint density, vessel length density, tortuosity, and generation-based branching metrics when VESGEN-style analysis is used. The biological interpretation is limited to quantitative vascular patterning and s

Live/dead viability and cytotoxicity staining assays are based on the simultaneous detection of intracellular esterase activity in metabolically active (viable) cells and membrane integrity loss in non-viable cells. In commonly used dual-staining approaches, membrane-permeant fluorogenic substrates are converted by intracellular esterases into fluorescent products in live cells, while impermeant DNA-binding dyes selectively enter cells with compromised plasma membranes and label nucleic acids in dead or dying cells, enabling discrimination between viable and non-viable populations by fluorescence microscopy or flow cytometry.

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.

Bromodeoxyuridine (BrdU) incorporation assay is based on the principle that BrdU, a thymidine analog, is incorporated into newly synthesized DNA during the S phase of the cell cycle, thereby serving as a marker of DNA replication and cellular proliferation. Incorporated BrdU can be detected using anti-BrdU antibodies following DNA denaturation, enabling visualization or quantification of proliferating cells through immunochemical detection methods such as immunofluorescence or immunohistochemistry.

ROS/oxidative-stress fluorescent staining uses cell-permeant fluorogenic probes that become fluorescent after oxidation inside cells or tissues; commonly used examples include DCFH-DA/DCFDA for broad cellular oxidant detection, DHE for superoxide-related signal detection, MitoSOX for mitochondrial superoxide-related signal detection, and CellROX probes for oxidative-stress-associated fluorescence readouts. The assay detects probe oxidation rather than a single ROS species unless the probe and analysis method have been chemically validated for that species. DCFH-DA enters cells, is deacetylated by intracellular esterases to DCFH, and produces fluorescent DCF after oxidation, so the readout is used as an operational measure of total cellular oxidative stress rather than a species-specific ROS measurement. DHE and MitoSOX can report superoxide-related oxidation, but red fluorescence alone can include non-specific ethidium-like oxidation products; HPLC or optimized spectral approaches are

Multiplex immunohistochemistry (mIHC), also known as tyramide dignal amplification (TSA), is an enzymatic detection method that uses horseradish peroxidase (HRP) to perform high-density in-situ labeling of target proteins or nucleic acids.

Cell-cycle analysis by flow cytometry measures DNA content in single cells to estimate the fraction of cells in G0/G1, S, and G2/M phases. Propidium iodide intercalates into DNA, and after RNA removal with RNase, fluorescence intensity reflects cellular DNA content: 2N cells are assigned to G0/G1, cells between 2N and 4N to S phase, and 4N cells to G2/M. DNA-content analysis alone cannot reliably separate G0 from G1 or G2 from M. Ki-67 can distinguish quiescent G0 cells from cycling cells, EdU or BrdU incorporation marks active DNA synthesis in S phase, and phospho-histone H3 staining identifies mitotic cells within the 4N population.

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

3D tumor spheroid invasion assay measures outward tumor-cell movement from a compact multicellular spheroid into a surrounding extracellular matrix, producing image-based readouts such as invasion area, invasion distance, cell dispersion, or time-resolved cell movement. The method models tumor-cell interaction with matrix components in three dimensions and is used to study invasive phenotypes in cancer models including glioblastoma, squamous cell carcinoma, breast cancer, prostate cancer, ovarian cancer, and other solid tumor systems.

Cancer immunology studies how the immune system recognizes, suppresses, edits, or fails to eliminate malignant cells through tumor antigen release, antigen presentation, T-cell priming, immune trafficking, tumor-cell killing, and feedback inhibition in the tumor microenvironment. The cancer-immunity cycle links tumor antigenicity, dendritic-cell priming, CD8+ T-cell infiltration, cytotoxic function, and immune-checkpoint regulation to tumor rejection or immune escape. Immune-checkpoint pathways such as PD-1/PD-L1 and CTLA-4 suppress antitumor T-cell activity and can be therapeutically blocked, but many tumors remain resistant because of poor antigen presentation, weak T-cell infiltration, suppressive myeloid cells, regulatory T cells, and tumor-intrinsic immune-exclusion programs. Unresolved questions include which immune-cell states predict response, how tumor-intrinsic pathways exclude immune cells, how myeloid suppression limits checkpoint blockade, and which combination strategies

Matrigel Transwell/Boyden chamber invasion assay measures the ability of cells to degrade or traverse an extracellular matrix-coated porous membrane and move from an upper chamber toward a chemoattractant in a lower chamber. Invasion is distinguished from migration by coating the membrane with Matrigel or basement membrane matrix; uncoated inserts measure migration, while coated inserts require cells to cross an ECM barrier before reaching the underside of the membrane.

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.

Invadopodia/fluorescent gelatin degradation assay detects proteolytic extracellular matrix degradation by cancer-cell invadopodia, which are actin-rich protrusive structures associated with matrix remodeling, invasion, and metastasis. The readout is generated by culturing cells on fluorescent gelatin and measuring dark degraded areas where fluorescent substrate has been locally removed, often together with immunofluorescent detection of invadopodia markers such as F-actin, cortactin, and TKS5.

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.