Apoptosis is a regulated, energy-dependent process of cell death characterized by distinct biochemical and morphological changes. No single assay captures all aspects, most experiments use a combination of methods targeting different hallmarks.
Caspase Activity Assays
Caspases are cysteine-aspartic proteases that execute the apoptotic program. Caspase-3 and -7 are the primary executioner caspases.
Fluorogenic substrates: a peptide recognition sequence (DEVD for caspase-3/7) linked to a fluorophore (AMC, AFC). Active caspase cleaves the peptide, releasing the fluorophore. Fluorescence (ex/em 355/460 nm for AMC) is measured in a plate reader. The assay is simple, quantitative, and suitable for high-throughput screening.
FRET-based reporters: cells expressing a DEVD peptide flanked by a fluorescent protein pair (e.g., CFP–DEVD–YFP) shift from FRET to donor emission upon caspase cleavage. These reporters allow real-time monitoring of apoptosis in live cells by microscopy or flow cytometry.
Annexin V / Propidium Iodide
In early apoptosis, phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. Annexin V binds PS with high affinity in a Ca²⁺-dependent manner. Conjugated to FITC, PE, or APC, it labels early apoptotic cells.
Propidium iodide (PI) is a membrane-impermeable DNA dye that enters only when the cell membrane is compromised (late apoptosis or necrosis). Dual staining distinguishes:
- Viable: Annexin V⁻/PI⁻
- Early apoptotic: Annexin V⁺/PI⁻
- Late apoptotic: Annexin V⁺/PI⁺
- Necrotic: Annexin V⁻/PI⁺
Analysis is performed by flow cytometry or fluorescence microscopy. The assay must be performed within 30 minutes of adding PI, as PI leaks from cells over time.
DNA Fragmentation
During apoptosis, endonucleases (CAD/DFF40) cleave DNA between nucleosomes, producing fragments in 180–200 bp multiples.
DNA laddering: genomic DNA is extracted, separated on an agarose gel, and stained with ethidium bromide. Apoptotic cells show a characteristic ladder pattern of 180 bp fragments. This is a qualitative, endpoint assay and is relatively insensitive, requires ~10⁶ cells.
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling): TdT incorporates labeled dUTP at the 3′-OH ends of fragmented DNA. The label (biotin, DIG, or a fluorophore) is detected by microscopy or flow cytometry. TUNEL labels all DNA breaks, including those in necrotic cells, so it is best combined with a morphology-based discrimination.
Mitochondrial Membrane Potential
Loss of mitochondrial membrane potential (ΔΨₘ) is a hallmark of the intrinsic apoptotic pathway. Fluorophores that accumulate in mitochondria based on their membrane potential are used:
- JC-1: emits green (monomer) at low ΔΨₘ and red (J-aggregates) at high ΔΨₘ. The red/green ratio indicates mitochondrial polarization.
- TMRE / TMRM: positively charged dyes that accumulate in polarized mitochondria. Loss of red fluorescence indicates depolarization.
The Right Controls
All apoptosis assays require positive (e.g., 1 µM staurosporine for 4–6 hours) and negative (untreated) controls. Assay timing matters: caspase activity peaks at 4–8 hours, PS exposure at 6–12 hours, and DNA fragmentation at 12–24 hours after the apoptotic stimulus.
