BenchMasteryMolecular biology techniques & virtual lab

Apoptosis assays

Live, unfixed cells are stained in calcium-containing buffer with fluorescent annexin V and a membrane-impermeant DNA dye such as PI or 7-AAD. Annexin⁺ dye⁻ cells are early apoptotic; double-positive cells are late apoptotic or necrotic.

% apoptoticSimulator readyCore simulatorEngine: Flow cytometry
Choose a sub-technique

Our ruleSimulations prepare you for the bench but don't replace hands-on practice.

Sub-technique you chose

Annexin V / PI

Live, unfixed cells are stained in calcium-containing buffer with fluorescent annexin V and a membrane-impermeant DNA dye such as PI or 7-AAD. Annexin⁺ dye⁻ cells are early apoptotic; double-positive cells are late apoptotic or necrotic.

How it differs from the standard method

  • Needs 1–2.5 mM Ca²⁺ throughout; avoid EDTA in the detachment step or wash it out.
  • Read within about an hour, unfixed and on ice.
  • Collect floating cells; use a gentle detachment for adherent cells.
  • Compensate FITC and PI with single-stained cells.
In this lesson · about 2 min read
  1. A sequence, not a state
  2. Why timing decides what you can say
  3. How the assays fail
  4. Key terms
  5. When to use it
  6. Sub-techniques

1A sequence, not a state

Apoptosis is an ordered programme, and each assay catches one step of it. Once a cell commits, initiator caspases activate the executioner caspases 3 and 7.

early

Caspase-3/7 activity

Fluorogenic DEVD substrates or antibodies to cleaved caspase-3. The earliest of the common readouts, and specific when a caspase inhibitor abolishes it.

early–mid

PS exposure

Annexin V binds PS in a Ca²⁺-dependent way. With a membrane-impermeant DNA dye it separates early apoptotic (annexin⁺ dye⁻) from late (annexin⁺ dye⁺) cells.

late

DNA fragmentation

TUNEL: terminal transferase adds labelled nucleotides to free 3′-OH ends. Works on fixed cells and tissue sections, but also labels necrotic and heavily damaged DNA.

2Why timing decides what you can say

Because each marker appears at a different point, the same culture gives different numbers at different times, and a single late time point can hide the route to death. Cells that are annexin⁺ PI⁺ may have been apoptotic hours ago or may have died by necrosis; only an earlier time point, when annexin⁺ PI⁻ cells dominate, shows that apoptosis came first.

3How the assays fail

  • Annexin V stained in PBS or with EDTA carried over from detachment: no calcium, no binding.
  • Scraping or harsh trypsinisation: healthy cells turn PI-positive.
  • Fixing before an annexin/PI read: every membrane becomes permeable.
  • No single-stained controls: FITC spillover into the PI channel moves early cells into the late quadrant.
  • TUNEL on ethanol-only fixed cells: the small fragments wash out, and apoptotic cells appear as a sub-G1 smear instead of TUNEL-positive.
  • No caspase-inhibitor or DNase controls: no proof that the signal means what it is supposed to.

Key terms

Practise these as flashcards →
Phosphatidylserine (PS)

A phospholipid normally kept on the inner leaflet; exposed on the outer leaflet early in apoptosis as an 'eat me' signal.

Annexin V

A protein that binds PS only in the presence of Ca²⁺; used fluorescently labelled.

Secondary necrosis

Loss of membrane integrity by apoptotic cells that are not cleared, as happens in culture.

DEVD

The tetrapeptide recognised by caspase-3 and -7, used in fluorogenic and luminogenic substrates.

Z-VAD-FMK

A cell-permeant, irreversible pan-caspase inhibitor; the specificity control for caspase signals.

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick-end labelling of DNA strand breaks.

Sub-G1

Cells with less than G1 DNA content in a DNA histogram; fragmented apoptotic nuclei that have lost DNA.

When to use it

  • To show that a treatment kills cells by apoptosis rather than necrosis or growth arrest, using an early marker and a time course.
  • To quantify apoptosis cell by cell in a mixed population by flow cytometry, or in tissue sections by TUNEL or cleaved caspase-3 staining.
  • To test whether a death pathway is caspase-dependent, with and without a caspase inhibitor.

Compared with alternatives

Viability & cytotoxicity →

Plate assays tell you how many cells are lost, faster and at higher throughput, but not how they died.

Proliferation & cell cycle →

A DNA-content histogram shows sub-G1 cells and whether survivors arrested in a phase.

Flow cytometry & FACS →

The instrument side: compensation, gating and controls that every flow apoptosis assay relies on.

Immunofluorescence →

Cleaved caspase-3 or TUNEL on fixed cells or sections when you need location and morphology.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Live, unfixed cells are stained in calcium-containing buffer with fluorescent annexin V and a membrane-impermeant DNA dye such as PI or 7-AAD. Annexin⁺ dye⁻ cells are early apoptotic; double-positive cells are late apoptotic or necrotic.

  • Needs 1–2.5 mM Ca²⁺ throughout; avoid EDTA in the detachment step or wash it out.
  • Read within about an hour, unfixed and on ice.
  • Collect floating cells; use a gentle detachment for adherent cells.
  • Compensate FITC and PI with single-stained cells.