MTT Assays

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molecular-bio
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beginner
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freemium
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video
INTRODUCTION
VIDEOS
MATERIALS
PROTOCOL
RESOURCES
DISCUSSION
Welcome! 

In this tutorial, we will cover MTT assays. MTT assays are quick, simple, and cheap assays that can be used to estimate proliferation and viability in cells under a multitude of conditions such as drug treatments, time, gene knockouts, etc. As such, they are a powerful tool in any lab. The videos in this tutorial will walk you through the theory, practice, and analysis of this assay. In addition, this tutorial contains a list of the reagents necessary to perform this experiment along with written out protocols, planning and analysis templates, and real-world examples.

Videos: 
  • Part 1: Theory
  • Part 2: Protocol
  • Part 3: Analysis
Experimental Details: 
  • Detailed experimental protocol
  • Materials list
Resources: 
  • Plate design templates
  • Cell counter template
  • Excel analysis template
  • Prism analysis template
  • Real-world examples [PRO]
  • Plate-design examples [PRO]

Preparation

  1. Prepare your MTT stock reagent. Note that MTT is soluble in water (10mg/mL), ethanol (20mg/mL), and dPBS or culture media (5mg/mL). We typically use a 5mg/mL solution in dPBS and mix by vortexing. Tip: This solution can be stored at 4°C for 3-5 days or at -20°C for approximately 6 months so you do not have to remake it each time you perform the assay.
  2. To make 10mL of MTT stock reagent, measure out 50mg (0.05g) of MTT powder into a 15mL falcon tube. Add 10mL of dPBS and mix thoroughly by vortexing and agitating until the mixture is thoroughly mixed and has no pellets or debris. Filter sterilize this solution and save it as the MTT stock reagent. It should appear bright yellow.
  3. Ensure that you have sterile dimethyl sulfoxide (DMSO) in the lab for the MTT solvent.
  4. Plate your cells exactly evenly (same # per well) and in replicates (4-8 minimum) into a 96-well-plate at least 24 hours in advance (longer if you have treatment conditions) and ensure they are properly attached and healthy before performing this assay. Tip: If treating cells for multiple days, plate them in the 96-well plate at the start of the treatment course at an appropriate density such that they can withstand treatment and grow for the period of the treatment without overgrowing the plate. At the final day of your treatment course, perform the MTT assay. In our experience, approximately 3000-5000 cells per well for moderately rapidly proliferating cells (doubling time ~24-36 hours) was sufficient to allow for 3 days of treatments followed by an MTT assay.

Assay

  1. Remove your cells from the incubator and check them carefully. The control cells should not be overgrown. They should be well attached with minimal debris and appear healthy. Tip: If you have a group that you expect to be highly proliferative, check that group as well. It is possible in some cases for the cells to overgrow and detach, thus resulting in a falsely decreased result with this assay. In this scenario, you will have to replate the cells with lower density per well and repeat the assay.
  2. Make your MTT working solution by diluting your MTT stock solution 1:10 in the cell culture media, making 110uL of working solution for each well you want to assay. Place this solution into a sterile reservoir. Tip: Calculate and make enough for all the plates you want to assay (and ideally a little bit extra). For example, if we were assaying one 96-well plate, we would calculate for 100 wells. For 100 wells, at 110 uL per well, we would need to make 11mL of MTT working solution. To do this, we would add 10mL of culture media and 1mL of MTT stock solution into a 15mL tube. Mix well and place into a sterile reservoir.
  3. In the tissue culture hood, remove the media from the plate. Tip: The best way to do this is to use a multichannel to completely remove the media from each well, working only from the corners of the wells to avoid dislodging cells and pipetting up and down 2-3 times to get rid of any debris. Wash the cells 1-2 times with sterile dPBS in the same volume used for culture media (~100-200 uL), removing completely each time.
  4. Once the cells are washed, remove the final dPBS from the plate completely. Add in the MTT working solution (i.e., MTT + media solution), using 110uL per well and dispensing with a multi-channel from a reservoir.
  5. Check the plate under the microscope to ensure cells are still attached. They should still appear healthy at this point although the media may seem slightly more orange. Place the plate back into the 37 degree incubator and incubate for 3-5 hours Tip: This can be left 24 hours if necessary
  6. After the time period has completed, remove the plate from the incubator and check the plate under the microscope. You should note the formation of crystals on the base of the plate and the cells should be gone. These are the formazan crystals you will be quantifying.
  7. CAREFULLY remove the media, pipetting only from the corners, and using only a multi-channel pipette to remove the media. Remove completely – any residual media will affect the precision of the assay moving forward.
  8. Resuspend the cells in 100uL of DMSO per well, pipetting aggressively up and down to resuspend the crystals completely. You should see the wells turn a deep purple in the most proliferative cells that you noted on your initial examination of the plate. Allow the plate to sit for 10 minutes (or up to 1 hour) before reading it out. Some gentle agitation on a plate rocker can help during this time.
  9. Read out the plate at OD=590 to get the absorbance values and save them into an excel sheet.

Analysis

Analysis is highly dependent on your assay question and conditions. However, for more information, please reference the video above, the examples provided, and the basic example shown in the downloadable protocol below.

Downloadable File

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