dGTP

The EdU incorporation assay measures DNA synthesis by adding the thymidine analog 5-ethynyl-2′-deoxyuridine to cells or tissues, where it is incorporated into newly synthesized DNA during S phase. Incorporated EdU is detected by copper-catalyzed azide-alkyne cycloaddition, in which a fluorescent azide covalently reacts with the ethynyl group on EdU, allowing S-phase cells to be detected by fluorescence microscopy, flow cytometry, or high-content imaging. EdU detection does not require DNA denaturation or anti-BrdU antibody access, which preserves sample structure and improves compatibility with immunostaining and multiparameter cytometry compared with BrdU-based detection. EdU can be cytotoxic in a cell-type- and exposure-dependent manner, so pulse duration, concentration, and continuous-labeling designs should be validated for each cell type.

PCNA immunodetection measures proliferative activity by detecting proliferating cell nuclear antigen, a nuclear protein associated with DNA polymerase δ function and DNA replication. The assay readout is the proportion of PCNA-positive nuclei among total counted cells, but PCNA labeling is not identical to BrdU labeling because PCNA can mark late G1/early S-associated replication competence and may persist beyond active DNA synthesis depending on fixation and extraction conditions.

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