MTT/MTS Proliferation Assay
Last updated: 5/22/16 By: Tim Starr
The purpose of this assay is to essentially measure the number of metabolically active cells in a 96-well plate. We currently use the CellTiter 96 AQueous One Solution Cell Proliferation Assay from Promega, although it is also possible to make up your own reagent.
- Number of cells: Count cells using trypan blue/hemocytometer. Place cells in a 96 well-plate, normally in 200 µl media. The number of cells per well depends on how fast the cells grow. Perform a titration test prior to the actual test by seeding 100 to 5000 cells in a 96 well plate, then observe confluency every 24 hours. In order to get an accurate measurement, cells should not have reached confluency by the end of the test (usually 5-7 days).
- Plate layout: Include at least 4 replicate wells per condition/cell line. Do not use the wells on the edges of the plate. Fill the cells on the edges of the 96 well plate (rows 1 & 12 and columns A & H) with media only and do not use as part of the experiment because these wells will have a significantly higher rate of evaporation. Prepare replicate plates for the number of time points you wish to collect, including a zero timepoint plate. Always remember to include a set of wells for media only and no treatment controls in your experimental design. You can include multiple timepoints on a single plate if there is room.
- Zero timepoint: Allow cells to setttle and adhere for ~4-6 hours in the incubator and take a reading (see below for how to take a reading). This reading will be subtracted from all subsequent readings in order to normalize to the actual number of cells seeded.
- MTS/MTT Absorbance Assay: At each timpoint, remove the 96 well plate from the incubator, add 20 µl of the Promega solution. This solution is a specially formulated tetrazolium salt, MTS, coupled to an electron coupling reagent, PES. Remove all bubbles from the wells. This can be accomplished by blowing air onto each bubble using a p1000 pipette, or any other method you can come up with. Return the plate to the 37ºC incubator and incubate for 2 hours. Promega claims you can incubate for anywhere from 1-4 hours, but whatever timepoint you choose, make sure to use the exact same incubation period for all readings. At the end of the incubation period read the absorbance using a spectrophotometer. Before putting the plate into your spectrophotometer, clean the bottom of the plate and make sure there aren't any scratches, fingerprints, or other marks. Remove the lid of the plate.
- Reading the absorbance: We use an EPOCH microplate spectrophotometer from BioTek using Gen5 software. For the CellTiter solution we read the absorbance at 490 nm, and optionally at 650 nm for a background reading. When setting up the Gen5 software you can use an Endpoint reading or an Area Scan. If using the Area Scan option, we think it is best to use a 3 x 3 grid with readings spaced at 1,200 microns x 1,200 microns. Based on our test readings, the edges of the well will give stronger and more variable readings than the center of the well, even when no cells are present, so by using the Area Scan you can reduce this variation.
- Calculating absorbance: If you take a 650 nm background reading, this needs to be subtracted well-by-well. In otherwords, subtact the 650 nm reading from well B2 from the 490 nm reading from well B2. Next, divide by the average zero timepoint reading for each well to normalize for starting cell numbers and display as "Absorbance (490 - 660) as percentage of day 0". Even though we try to start with the same number of cells in every well, we have found that when counting different types of cells or different conditions, the number of plated cells can vary considerably even after counting with the hemocytometer. At this point you will have a absorbance value as a percentage of initial absorbance value. Use these numbers to calculate Standard Error of the Mean (SEM) and perform a T.Test comparing each sample to the control.
- Graphing the results: There are two options for graphing the results. The first option is to graph the average absorbance as percent of day0 0 values (usually this is from 100 to 300%). The second option is to graph the experimental conditions/cells as a percentage of the control condition/cell, which will put all the control readings for every day at 100%.