The MTS assay is a colorimetric method used to measure cell viability, proliferation, and cytotoxicity by detecting the bioreduction of a tetrazolium compound into a water-soluble formazan dye directly in the culture medium.
What does MTT stand for?
MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium salt widely used in colorimetric assays to assess cell metabolic activity.
You’ll often see the name shortened to just “MTT,” but that mouthful is what it actually stands for. The chemical name describes its structure—a thiazolyl blue tetrazolium bromide compound. Living cells reduce MTT into purple formazan crystals, which you can then measure with a plate reader. According to the National Institutes of Health (NIH), MTT remains a gold standard in cell viability studies, even though newer options have come along.
How do MTT and MTS assays differ?
MTT requires an extra solubilization step to dissolve the formazan crystals, while MTS produces a water-soluble formazan that doesn’t need further processing.
That difference makes MTS way easier to run. After incubation, you just read the plate—no DMSO or other solvent needed. MTS reagents (like CellTiter 96® AQueous One Solution) are also gentler on cells, so you can incubate longer without killing them off. A Promega technical guide calls MTS a top pick for high-throughput screening because it’s simple and plays nice with automation.
What kind of assay is MTT?
The MTT assay is a colorimetric cell viability test that measures metabolic activity by tracking how cells reduce MTT into formazan.
It’s part of the tetrazolium reduction family and shows up everywhere—cytotoxicity tests, drug screens, apoptosis studies. The purple formazan correlates with how many metabolically active cells you’ve got, though it won’t tell you if cells are proliferating or just cranking up their metabolism. Thermo Fisher Scientific notes that MTT is cheap and reliable, but newer dyes like resazurin can be more sensitive.
Does MTS harm cells?
MTS is far gentler on cells than MTT, so you can run longer experiments or take repeated measurements without worrying about killing your cultures.
That’s a big advantage if you’re tracking viability over time. The water-soluble formazan also means no organic solvents, which keeps cell stress low. Agilent’s MTS protocol points out that MTS works well even with sensitive primary cells, since it barely impacts their health.
How do you calculate MTS assay results?
Start by subtracting blank absorbance, then divide treated samples by controls and multiply by 100 to get percent viability:
- Subtract the absorbance of blank wells (media only) from all sample wells.
- Divide the corrected absorbance of treated wells by the average corrected absorbance of control (untreated) wells.
- Multiply by 100 to convert to percent viability: Viability (%) = (Sample Abs – Blank Abs) ÷ (Control Abs – Blank Abs) × 100.
This assumes the formazan dye scales linearly with viable cell number. For solid data, always run controls in triplicate and keep incubation times identical. Bio-Rad’s MTS protocol suggests making a standard curve with known cell numbers to confirm your results.
Can cell viability go above 100%?
Yes, viability can top 100% when treatments boost metabolism or cell growth beyond control levels.
It’s rare, but you might see this with growth factors, mitogens, or low-toxicity stimulants that rev up metabolism or cell division. On the flip side, cytotoxic agents push viability below 100%. Abcam’s MTS protocol warns that values over 100% could mean assay quirks or nonlinear responses at high cell densities. Always include proper controls and double-check with a backup method like trypan blue if you get weird numbers.
How do you run an MTT assay?
Incubate cells with MTT for 2–4 hours, dissolve the formazan with DMSO, then measure absorbance at 570 nm.
- Remove culture media and add serum-free media with 10% MTT solution (final concentration ~0.5 mg/mL).
- Incubate plates at 37°C in a humidified 5% CO₂ incubator for 2–4 hours.
- Carefully remove the media and add DMSO to dissolve formazan crystals (100–200 μL per well).
- Incubate 10–15 minutes on a shaker to ensure complete dissolution.
- Measure absorbance at 570 nm (reference: 630–690 nm) using a microplate reader.
MilliporeSigma’s MTT protocol stresses keeping plates in the dark while formazan develops and using DMSO-treated wells as blanks to correct background.
Why use DMSO in an MTT assay?
DMSO dissolves the insoluble purple formazan crystals so you can measure absorbance and quantify cell viability.
Formazan forms inside live cells during MTT reduction but won’t dissolve in water. DMSO breaks open cell membranes and solubilizes the crystals, releasing the dye for accurate readings. Thermo Fisher’s MTT manual recommends using sterile, cell culture-grade DMSO and warns that leftover crystals can mess with your data. Add DMSO only after removing MTT-containing media to avoid lysing cells before formazan forms.
What does IC50 mean in an MTT assay?
IC50 is the compound concentration that cuts cell viability in half compared to untreated controls.
You find it by plotting viability against a range of doses and finding where the curve hits 50%. Drug developers love IC50—it tells them how potent and toxic a compound is. GraphPad Prism’s guide suggests using nonlinear regression (log(inhibitor) vs. normalized response) for reliable IC50s. Just remember: cell type, assay length, and starting cell density all tweak the number.
What’s a good positive control for an MTT assay?
A positive control is a known cytotoxic agent—like staurosporine or hydrogen peroxide—that reliably slashes viability to a predictable low level.
It’s your way of checking that the assay is working and the detection system isn’t broken. Common picks include 1–10 μM staurosporine for apoptosis or 0.1% Triton X-100 for full lysis. Roche’s Cell Proliferation Kit protocol says to include positive controls on every plate and compare treated samples to both positive and negative controls (untreated cells). That way, you can trust your viability numbers.
Who invented the MTT assay?
Tim Mosmann first described the MTT assay in 1983 in the Journal of Immunological Methods.
His paper introduced MTT as a fast, cheap way to measure proliferation and viability in mouse cell lines. Later tweaks by Denizot and Lang (1986) made the method work for more cell types. The Journal of Immunology notes that Mosmann’s work revolutionized immune cell testing and set the stage for modern cytotoxicity assays.
Who developed the MTT assay?
Tim Mosmann developed and published the MTT assay in 1983 while at the University of Alberta.
His colorimetric method replaced clunky radioactive assays and became a lab staple in immunology, cancer research, and toxicology. Nature Protocols calls it one of the most cited protocols in cell biology, with over 20,000 citations to date.
What exactly is the MTS reagent?
The MTS reagent is a tetrazolium compound that live cells reduce into a soluble formazan dye right in the culture medium.
You’ll see it sold under names like CellTiter 96® AQueous One Solution (Promega) or MTS Cell Proliferation Assay (ATCC). Unlike MTT, it skips the solubilization step, so your workflow is cleaner. Promega’s product page says MTS fits high-throughput plates and drug discovery screens like a glove.
Is MTT really the best assay to use?
MTT is reliable, but it’s not always the best choice—it’s cytotoxic, leaves insoluble waste, and isn’t as sensitive as MTS or resazurin.
You’ll need DMSO to dissolve formazan, and some test compounds can mess with the readout. Bio-Rad’s viability guide recommends picking an assay based on your goals: go with MTT for simplicity, MTS for convenience, or resazurin for sensitivity and continuous monitoring. Think about your cell type, how long the assay runs, and what you’ll do next.
How can I make my MTT assay better?
Use fresh, filter-sterilized MTT at 0.5 mg/mL, optimize incubation to 2–4 hours, and fully dissolve formazan with DMSO before reading.
Stick to consistent plating densities and run plenty of replicates for solid stats. Thermo Fisher’s optimization guide suggests pre-warming reagents to 37°C and keeping plates in the dark while formazan develops. For long experiments, try phenol red-free media to cut background. Always include controls and, if you can, validate with a second assay.
