HATC

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.

Suspension spheroid formation by low-adhesion or forced aggregation is a scaffold-free 3D culture method in which cells are prevented from attaching to plastic and are guided to interact with each other, forming compact multicellular aggregates through cell-cell adhesion, gravity-driven settling, microwell confinement, or centrifugation-assisted aggregation. The method detects the capacity of a cell population to self-assemble into spheroids, and the main readouts are spheroid formation efficiency, morphology, compactness, projected area or diameter, circularity, viability, proliferation, and experimental responses such as drug sensitivity. Classic implementations include hanging drops, agarose or hydrogel microwells, ultra-low-attachment round-bottom wells, and centrifugation-assisted aggregation in non-adherent wells. Low-adhesion culture shifts the system away from cell-substrate adhesion and toward cell-cell adhesion, while round-bottom or microwell geometry concentrates cells into

Hanging drop spheroid culture is a scaffold-free 3D culture method in which a small droplet of cell suspension is inverted so that suspended cells sediment by gravity toward the lowest point of the drop, aggregate, and form a multicellular spheroid with direct cell-cell contact. Spheroids generated by this method are used to study 3D cell cohesion, cell-ECM interactions, drug response, co-culture organization, and tumor-like microenvironmental behavior. The primary readouts are spheroid formation efficiency, spheroid size, circularity or compactness, viability, and treatment response; these can be measured by bright-field microscopy, fluorescence viability staining, ATP-, fluorescence-, or colorimetric-based assays, and image-based diameter or volume calculations.

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,

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