Sean Lauber:MTT Cell Proliferation Assay: Difference between revisions

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The following protocol has been optimized for growing lewis lung carcinoma (LLC) cells over a 3 day period. If you're working with a suspension or adherent/suspension cell line (like LLC cells) then you should not remove the supernatant when adding the MTT reagents. If you want to recover supernatant, it's best to seed additional wells for this purpose.
The following protocol has been optimized for growing lewis lung carcinoma (LLC) cells over a 3 day period. If you're working with a suspension or adherent/suspension cell line (like LLC cells) then you should not remove the supernatant when adding the MTT reagents. If you want to recover supernatant, it's best to seed additional wells for this purpose.


1. Grow cells up and split 1:2 when confluent (I like to passage them at least once before treatment)
1. Seed at an appropriate density in media and allow the cells to adhere to the plate for 2 hours


2. After 24 hours, trypsinize these cells and seed at an appropriate density in PBS (1000 cells per 100 ul on a 96-well plate has worked well for me) and allow the cells to adhere to the plate for 2 hours
2. Replace with media containing whatever you're studying with 1% FBS (final volume = 100 ul)


3. Replace with media containing whatever you're studying with 1% FBS (final volume = 100 ul)
3. Incubate the cells at 24-72 hours at 37°C/5% CO2.
 
5. Incubate the cells at 24-72 hours at 37°C/5% CO2.
   
   
6. Add 20 ul of 5 mg/ml MTT (ensure that the MTT is warmed and completely dissolved) directly to the well (final vol = 120 ul).  
4. Add 20 ul of 5 mg/ml MTT (ensure that the MTT is warmed and completely dissolved) directly to the well (final vol = 120 ul).  


7. Incubate for 4 hours at 37°C in the dark.
5. Incubate for 4 hours at 37°C in the dark.


8. Add 120 ul of the MTT solvent solution (warmed).
6. Add 120 ul of the MTT solvent solution (warmed).


9. Pipette up and down vigorously to lyse the cells and dissolve the colorized product. If you look at the bottom of the plate and notice specks of purple, then you haven't pippeted well enough. Everything should dissolve. If it doesn't dissolve, put it on a shaker for 15 minutes (in the dark), and then try again.
7. Pipette up and down vigorously to lyse the cells and dissolve the colorized product. If you look at the bottom of the plate and notice specks of purple, then you haven't pippeted well enough. Everything should dissolve. If it doesn't dissolve, put it on a shaker for 15 minutes (in the dark), and then try again.


10. Read at 550 nm.
8. Read at 550 nm.




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NOTE!! In some cases the cells won't lyse in this solvent. LLC cells lyse fine but B16 cells did not. In this situation you may want to use a different solvent (straight DMSO at 200 ul after removing the supernatant should work). Alternatively, waiting overnight for the cells to lyse may work with the proposed solvent.
NOTE!! In some cases the cells won't lyse in this solvent. LLC cells lyse fine but B16 cells did not. In this situation you may want to use a different solvent (straight DMSO at 200 ul after removing the supernatant should work). Alternatively, waiting overnight for the cells to lyse may work with the proposed solvent.
  MTT solution:
  50 mg MTT
  10 ml PBS
  vortex vigorously, then filter sterilize
  store in 1 ml aliquots at -20*C

Latest revision as of 13:56, 5 November 2013

The following protocol has been optimized for growing lewis lung carcinoma (LLC) cells over a 3 day period. If you're working with a suspension or adherent/suspension cell line (like LLC cells) then you should not remove the supernatant when adding the MTT reagents. If you want to recover supernatant, it's best to seed additional wells for this purpose.

1. Seed at an appropriate density in media and allow the cells to adhere to the plate for 2 hours

2. Replace with media containing whatever you're studying with 1% FBS (final volume = 100 ul)

3. Incubate the cells at 24-72 hours at 37°C/5% CO2.

4. Add 20 ul of 5 mg/ml MTT (ensure that the MTT is warmed and completely dissolved) directly to the well (final vol = 120 ul).

5. Incubate for 4 hours at 37°C in the dark.

6. Add 120 ul of the MTT solvent solution (warmed).

7. Pipette up and down vigorously to lyse the cells and dissolve the colorized product. If you look at the bottom of the plate and notice specks of purple, then you haven't pippeted well enough. Everything should dissolve. If it doesn't dissolve, put it on a shaker for 15 minutes (in the dark), and then try again.

8. Read at 550 nm.


You might find it useful to include a standard curve. For this I would plate 500, 1000, 5000, 10000, 30000, 70000, 100000 cells shortly before treating with MTT (allowing time for adherence). However this is unnecessary since you're probably going to be expressing it as a % growth from untreated cells (untreated cells = 100%)

In one situation I had too many cells and the absorbance was out of the detectable range. In this case you can dilute the solution with some amount of PBS and re-read the absorbances (1:2, 1:4, etc until it's on scale). Be sure to dilute all samples if you want to be able to compare them.

If you have an adherent population, feel free to remove the supernatant for ELISAs. Then simply add 100 ul of media on top of the cells before adding the 20 ul of MTT reagent. 

 MTT solvent:
 8 mM HCl
 0.2% IGEPAL CA-630 (in isopropanol)


NOTE!! In some cases the cells won't lyse in this solvent. LLC cells lyse fine but B16 cells did not. In this situation you may want to use a different solvent (straight DMSO at 200 ul after removing the supernatant should work). Alternatively, waiting overnight for the cells to lyse may work with the proposed solvent. 

 MTT solution:
 50 mg MTT
 10 ml PBS
 vortex vigorously, then filter sterilize
 store in 1 ml aliquots at -20*C
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