ML-20

Mitochondrial membrane potential fluorescent assays estimate ΔΨm in living cells using lipophilic cationic dyes such as TMRM, TMRE, rhodamine 123, and JC-1, which accumulate in mitochondria according to membrane polarization; loss of signal after FCCP or CCCP treatment is interpreted as mitochondrial depolarization. TMRM/TMRE and rhodamine 123 are commonly used for semi-quantitative live-cell microscopy or flow cytometry, while JC-1 can report a shift from red aggregate fluorescence to green monomer fluorescence during depolarization; interpretation requires controls because dye concentration, quenching mode, cell type, dye efflux, and mitochondrial mass can affect fluorescence independently of ΔΨm.

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.

A method of simulating the in vivo environment in vitro to maintain the cell growth, differentation and main functions.

The CCK-8/WST-8 assay is based on the reduction of the water-soluble tetrazolium salt WST-8 to a water-soluble formazan product by cellular dehydrogenases in metabolically active cells, where the generated formazan amount is proportional to the number of living cells and is quantified by measuring absorbance in the visible range, providing a colorimetric readout for cell viability and proliferation assessment. This class of tetrazolium-based assays improves upon earlier MTT-based systems by producing a water-soluble formazan, eliminating the need for organic solubilization steps and enabling direct spectrophotometric measurement in culture medium.

Cell counting-based growth curve assays quantify cell proliferation by directly measuring changes in viable cell number over time using manual or automated counting methods such as hemocytometer-based counting or instrument-assisted cell enumeration, enabling construction of growth curves that reflect population expansion dynamics in response to culture conditions. A widely used approach is trypan blue exclusion with hemocytometer counting, where membrane-compromised (non-viable) cells take up the dye, allowing discrimination between viable and non-viable cells while simultaneously enabling total cell number quantification. Repeated sampling across time points allows estimation of proliferation rate, growth phases, and comparative growth kinetics between experimental conditions.

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.

Cell counting and cell-density analysis estimate the number of cells in a known volume or field area. Manual hemocytometer counting uses a chamber of defined geometry to convert counted cells into cells/mL, while automated counters and image-analysis workflows detect cell objects from optical, brightfield, fluorescence, impedance, or digital-image features. Trypan blue viability counting is based on dye exclusion: viable cells with intact membranes exclude dye, while non-viable cells with compromised membranes stain blue. The readout is total cell density, viable-cell density, dead-cell density, and percent viability. Cell density can also be estimated from microscopy images by counting objects per image area, from flow cytometry using calibrated volume or reference particles, or from in situ microscopy in bioreactors after calibration against reference methods such as hemocytometer or flow cytometry.

Fluorescent plasma-membrane potential dye assays measure changes in cell membrane potential using voltage-sensitive dyes whose fluorescence changes when cells depolarize or hyperpolarize. Anionic bis-oxonol dyes such as DiBAC4(3) enter depolarized cells more readily and show increased fluorescence after intracellular binding, while hyperpolarization reduces dye accumulation and fluorescence. FMP/FLIPR membrane-potential dyes are used for faster, homogeneous microplate assays of ion-channel or receptor-mediated membrane-potential changes.

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.

Mitochondrial membrane potential staining measures the electrochemical polarization across the mitochondrial inner membrane in live cells using lipophilic cationic fluorescent probes; early rhodamine-based work showed that selective mitochondrial dye accumulation is lost when the mitochondrial transmembrane potential is dissipated. JC-1 reports mitochondrial polarization by shifting from green monomer fluorescence to red J-aggregate fluorescence as dye concentration increases within energized mitochondria; therefore, the red/green fluorescence ratio is used as a relative readout of mitochondrial membrane potential. TMRE or TMRM staining provides a single-channel relative readout because these cationic rhodamine esters accumulate in polarized mitochondria, and lower fluorescence indicates reduced mitochondrial polarization when acquisition and dye-loading conditions are controlled. Mitochondrial mass staining is commonly performed with MitoTracker Green FM or related MitoTracker dyes as

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

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,

The MTT assay is a colorimetric endpoint assay for estimating viable cell number, cell growth, cytotoxicity, or cell activation in cultured mammalian cells. Living cells reduce the yellow tetrazolium salt MTT into purple/blue formazan, while dead cells do not generate the same signal; the resulting color can be quantified with a multiwell spectrophotometer. MTT reduction is commonly interpreted as a readout of metabolic activity that often correlates with viable cell number, but it should not be treated as a direct cell-counting method unless the assay is optimized for the cell type and experimental condition. Studies show that MTT reduction can involve mitochondrial and non-mitochondrial reducing systems, and formazan may accumulate in intracellular lipid droplets rather than simply marking mitochondria.

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