MTT vs CCK-8 and other cell viability assays, plus apoptosis and cell cycle analysis

No plate assay counts cells. Each measures something that live cells have and dead ones lose, and assumes that signal tracks cell number:

• Metabolic activity: MTT, CCK-8 and resazurin rely on cellular reductases, mostly NAD(P)H-dependent, reducing a dye.
• Membrane integrity: dyes such as propidium iodide enter only cells with damaged membranes; LDH release assays measure enzyme leaking out of them.
• ATP: luciferase light output tracks ATP, which falls quickly when cells die.

A drug that slows metabolism without killing cells will lower an MTT reading but not a dead-cell stain. When results disagree, the assays are usually measuring different things correctly.

The outline shows the logic. Follow the volumes in your kit or reagent sheet.

1. Seed cells in a 96-well plate and let them attach, usually overnight.
2. Treat with compounds for the planned time, with vehicle and blank-medium controls.
3. Add MTT to a final concentration of about 0.5 mg/mL.
4. Incubate 2–4 hours at 37 °C until purple crystals are visible in the cells.
5. Remove the medium carefully and dissolve the formazan in DMSO, or add an SDS solubilising solution.
6. Shake gently, then read at 570 nm with a reference wavelength of 630–690 nm.

CCK-8 uses WST-8, a tetrazolium that cells reduce outside the cell with the help of an electron carrier. The orange formazan dissolves in the medium, so there is no solubilisation step: add the reagent, incubate for one to four hours and read at 450 nm. Because nothing is removed, CCK-8 has fewer handling errors than MTT, lower toxicity, and lets you read the same plate more than once. It costs more per well, which is why MTT remains common for large, simple screens.

Acridine orange (AO) enters every cell and stains nucleic acids green. Ethidium bromide (EB) or propidium iodide (PI) enters only cells whose membranes have failed, and stains them orange to red. Viewed together under a fluorescence microscope:

AO also accumulates in acidic compartments and fluoresces red there, which makes it useful for studying lysosomes and autophagy. Count several fields per sample, because the method is semi-quantitative.

PI binds DNA in proportion to its amount, so flow cytometry of PI-stained cells separates them by DNA content: cells in G0/G1 have one copy, cells in G2/M have two, and S-phase cells sit between them.

1. Harvest cells, including floating cells, and wash in PBS.
2. Fix by adding cold 70% ethanol drop by drop while vortexing gently, then hold at 4 °C or -20 °C.
3. Wash out the ethanol and resuspend in PI staining solution containing RNase A, because PI also binds RNA.
4. Incubate in the dark, then acquire on a flow cytometer with doublet discrimination.
5. Model G0/G1, S and G2/M fractions with cell cycle software.

A peak below G0/G1, the sub-G1 population, contains fragmented DNA and is often read as apoptotic cells.

A viability result says that cells responded; other assays say how. Mitochondrial function is covered in the SDH activity guide, enzyme readouts in the enzyme activity assay guide, and protein markers of apoptosis such as cleaved caspase-3 need validated antibodies. MTT reagent, CCK-8, AO staining and cell cycle kits are in the biochemical and cell-based assay range.