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Short answer
The XTT assay is a colorimetric test of cell viability based on metabolic activity. Living cells reduce the water-soluble tetrazolium salt XTT to an orange formazan product, and a plate reader measures that colour. Because the product dissolves in the culture medium, no lysis or solubilization step is needed. The exact incubation time, reagent ratio and reading wavelengths depend on the kit, so follow the insert for the product in your lab.
Definition and scope
XTT is a tetrazolium salt used to estimate how many metabolically active cells are in a culture. Thermo Fisher describes its CyQUANT XTT assay as a colorimetric assay that assesses cell viability “as a function of cell number based on metabolic activity”, with the water-soluble XTT converted to an orange-coloured formazan product. ReprocELL’s protocol for 96-well plates describes the same reaction: reduction of XTT produces an orange formazan derivative, and the colour change happens in the culture medium rather than inside the cells.
That makes it a relative measure. A higher absorbance means more metabolic activity in the well, which tracks cell number only within the range where you have shown the two are proportional (see the checklist below). It does not count cells, and it does not by itself distinguish a dead cell from one that is alive but metabolically quiet. For direct counting see the hemocytometer guide; for a different metabolic readout see the ATP assay guide.
How the XTT assay is run in practice
This section describes the general sequence in two vendor protocols; it is not a replacement for the instructions packaged with your reagent.
- Prepare the working reagent just before use. XTT is combined with a separate activation or electron coupling reagent. Thermo Fisher says to use the working solution promptly after it is prepared.
- Add it to cells in culture. The working solution is added to wells that already hold cells in medium. No wash is required after staining in the Thermo Fisher protocol.
- Incubate. Plates go back into a CO2 incubator at 37 °C for the time the protocol specifies.
- Read absorbance. Orange formazan is read near 450 nm. ReprocELL adds a reading at 630-690 nm for non-specific absorbance, while Thermo Fisher lists a second reading at 660 nm without stating its purpose; follow the readings your protocol names.
Reading needs a plate reader that covers the wavelengths your protocol names; see What Is a Plate Reader?.
Two vendor protocols compared
The parameters below come from two published vendor protocols, checked on 2026-10-29. They differ on almost every setting, which is the practical point: the numbers belong to the kit, not to “the XTT assay”.
| Parameter | ReprocELL D040 (96-well, Alvetex Scaffold) | Thermo Fisher CyQUANT XTT |
|---|---|---|
| Activation step | 0.1 mL Activation Reagent per 5.0 mL XTT reagent (per ATCC manufacturer instructions) | 1 mL Electron Coupling Reagent per 6 mL XTT Reagent, vortexed |
| Added per well | 50 µL activated XTT to 100 µL medium (2:1 medium to activated XTT) | 70 µL working solution per well |
| Incubation | 1-2 hours at 37 °C, CO2 appropriate to the cell type | 4 hours at 37 °C in a CO2 incubator |
| Absorbance reading | 450-500 nm; non-specific absorbance at 630-690 nm | 450 nm and 660 nm |
| Blank | Well with no cells: 100 µL medium plus 50 µL activated XTT | At least three wells with complete culture medium and no cells |
ReprocELL gives the calculation as: specific absorbance = A450 (sample) − A450 (blank) − A630 (sample), and labels its protocol as an example that may need further optimisation.
Controls and set-up checklist
Every item below is taken from the two protocols above.
- No-cell blank. Include medium-plus-reagent wells with no cells and subtract them. Thermo Fisher specifies at least three.
- Titrate cell number. Thermo Fisher describes titrating roughly 103 to 105 cells per well for a standard curve. ReprocELL advises establishing the range where cell number and signal are linear for each cell type; its example was linear up to 50,000 HepG2 cells per well.
- Prepare the working solution fresh. Activate immediately before use rather than in advance.
- Read at the wavelengths your protocol names. ReprocELL reads 630-690 nm for non-specific absorbance; Thermo Fisher lists a second reading at 660 nm without stating its purpose.
- Protect the reagent. Thermo Fisher notes that freeze/thaw affects performance and that single-use vials are kept at -20 °C.
- Do not use it for imaging. The Thermo Fisher protocol states the assay is not for fluorescence microscopy.
For vessel areas, media volumes and seeding densities, see the cell culture reference numbers guide.
Where it matters for labs
For a lab comparing viability methods, the useful distinction is what each one reads. The MTT assay is also tetrazolium-based, but MTT’s formazan needs a separate solubilization step, as that guide explains; XTT’s orange formazan is soluble in the medium, so no solubilization step is needed. The ATP assay uses a different principle again. Which fits depends on the wavelengths your plate reader covers and the incubation time your protocol specifies.
For anyone specifying consumables and equipment, the practical points are modest and come straight from the protocols: a plate reader able to read around 450 nm plus the second wavelength your kit lists, a CO2 incubator for the incubation step (see What Is a Lab Incubator?), and the kit-specific activation or coupling reagent supplied with the XTT reagent.
Common misunderstandings
“XTT counts live cells.”
It reports metabolic activity. The Thermo Fisher protocol itself frames the result as viability “as a function of cell number”, which holds only inside a verified linear range.
“Any XTT protocol can be used with any XTT reagent.”
The table shows that activation ratios, incubation times and read wavelengths differ between two published protocols. Use the parameters of the reagent you actually have.
“The 450 nm reading is enough.”
Both protocols list a second, longer-wavelength reading alongside 450 nm (630-690 nm in ReprocELL, 660 nm in Thermo Fisher) and use a no-cell blank. ReprocELL states that its 630-690 nm reading is for non-specific absorbance; Thermo Fisher does not state the purpose of 660 nm.
FAQ
What does the XTT assay measure?
Metabolic activity of cells in culture, read as the orange formazan produced when cells reduce XTT. It is used as a proxy for viable cell number.
Why does the XTT assay not need a solubilization step?
The formazan product of XTT is water-soluble and forms in the culture medium, so the colour can be read directly from the plate.
What wavelength is the XTT assay read at?
Both protocols above read at 450 nm, with a second reading at a longer wavelength. ReprocELL reads 630-690 nm for non-specific absorbance; Thermo Fisher lists 660 nm without stating its purpose.
How long is the incubation?
It depends on the kit: 1-2 hours in the ReprocELL protocol and 4 hours in the Thermo Fisher protocol.
Sources
- ReprocELL, protocol D040, “XTT Cell Viability Assay for Alvetex Scaffold 96 Well Plate Format”: reprocell.com (checked 2026-10-29).
- Thermo Fisher Scientific, “CyQUANT XTT Cell Viability Assay Protocol”: thermofisher.com (checked 2026-10-29).
