Apoptosis Assays
Apoptosis (programmed cell death) is characterized by specific morphological and biochemical changes. Several complementary assays distinguish apoptosis from necrosis and identify the stage of cell death.
Annexin V / Propidium Iodide staining is the most common flow cytometry–based apoptosis assay. During early apoptosis, phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. Annexin V, a PS-binding protein conjugated to a fluorophore (FITC, APC, PE), binds to exposed PS. Propidium iodide (PI) or 7-AAD enters only cells with compromised membranes (late apoptosis/necrosis).
- Annexin V⁻ / PI⁻: live cells
- Annexin V⁺ / PI⁻: early apoptotic cells
- Annexin V⁺ / PI⁺: late apoptotic / necrotic cells
TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labeling) detects DNA fragmentation, a hallmark of late apoptosis. Terminal deoxynucleotidyl transferase (TdT) adds labeled dUTP to the 3′-OH ends of fragmented DNA. Detection is by fluorescence microscopy or flow cytometry.
Caspase assays measure the activity of caspases, the executioner proteases of apoptosis. Fluorogenic substrates (e.g., DEVD-AMC for caspase-3/7) are cleaved by active caspases, releasing a fluorescent signal. Caspase-3/7 activity is the most widely used biochemical marker of apoptosis.
Cell Cycle Analysis
Cell cycle analysis measures the distribution of cells across G₀/G₁, S, G₂, and M phases, revealing the effects of drugs, nutrients, or genetic changes on cell division.
DNA content analysis by flow cytometry uses a fluorescent DNA-binding dye (PI, DAPI, Hoechst 33342). Fixed and permeabilized cells are stained, and the fluorescence intensity is measured. The intensity is proportional to DNA content:
- G₀/G₁: 2N DNA content (one peak)
- S: between 2N and 4N (the region between peaks)
- G₂/M: 4N DNA content (second peak)
The percentage of cells in each phase is calculated using modeling software (ModFit, FlowJo Watson algorithm).
BrdU/EdU pulse-chase provides more detailed cell cycle information. A short pulse of BrdU or EdU labels cells in S-phase. After a chase period, cells are analyzed for both DNA content and BrdU/EdU incorporation, revealing the rate of S-phase progression and the duration of each phase.
Phospho-histone H3 (Ser10) staining specifically marks mitotic cells, providing a G₂/M phase marker that distinguishes G₂ from M by flow cytometry or microscopy.
