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College of Life Sciences Microbiology and Molecular Biology
Tessem Laboratory - Diabetes Research Group

MTT Assay

Background Info: MTT

The MTT assay is a sensitive and reliable indicator of the cellular metabolic activity and is preferred over the other methods measuring this end-point like the ATP and 3H-thymidine incorporation assay, the latter employing radioactivity . The assay relies on the reduction of MTT, a yellow water-soluble tetrazolium dye, primarily by the mitochondrial dehydrogenases, to purple colored formazan crystals. The formazan product is analyzed spectrophotometrically (570 nm) after dissolution in DMSO, the spectra of nanoparticle-treated and untreated cells giving an estimate of the extent of cytotoxicity

Things to know before starting:

a. Thiazolyl blue tetrazolium bromide is light sensitive, so it must be stored in the dark (can last in the fridge up to 4 weeks in PBS) and lights should be kept off of it directly.

Preparation of 12mM MTT/PBS Solution:

Add 1mL of PBS to every 5mg thiazolyl blue tetrazolium bromide(MTT).

  1. Mix by vortexing or sonication until dissolved
  2. Once prepared, the MTT solution can be stored for 4 weeks at 4 degrees Celsius, protected from light

Step 1: Plate cells

Plate cells in 96-well plates. As you do so, make sure to leave room for the following on each plate:

1. One column left COMPLETELY EMPTY (no cells or media). This will be used to measure background absorbance during data quantification.

2. One column with NO CELLS. This column will be filled with the Media/MTT mixture after treatment has occured.

Protocol for GLT and Cytokine treatments:

  1. Mix 1mL 12mM MTT/PBS solution with 9mL media(per plate)
  2. Aspirate(dump into a paper towel) old media from plate
  3. Add 100ul per well of the MTT/Media solution to each well(skip row 12) of a 96-well plate.(Light sensitive)
  4. Incubate for 3 hours.
  5. Aspirate off, rinse with 100uL of PBS
  6. Add 50uL of DMSO to each well
  7. Cover with tin foil(important on this step, otherwise your top plate will get ruined) and place on shaker for 20min

  8. Take to the plate reader in Berges/Poole Lab

    1. Turn Plate Reader on(button)
    2. Log into NIVO software(instructions on wall)
    3. Select MTT protocol, read at 570nm
    4. Select Export, save as an excel
  9. If Berges Lab plate reader is not accessible, please locate your nearest grad student(or senior undergrad) in order to use a different one.

  10. Analyze

    1. Copy and paste raw data from wells into prism, run an outlier test.
    2. Take cleaned data from each plate, paste into new excel
    3. Take the average of each of the columns, then subtract the negative control column(usually 11, has no cells in it)
    4. Average the SCC(standard cell culture) controls and divide by that value to normalize to the SCC
    5. Export values to prism to run outlier and anova significance tests