Need advice on cytotoxicity assay - (Jul/15/2008 )
Hi all, I'm curently doing a proliferation assay to test some plant extract's ability to inhibit cell proliferation and i have chosen the MTT assay for that. However, how do i show that my extracts are actually inhibiting proliferation instead of inducing cytotoxicity ? Could someone advice me on a suitable cytotoxicity assay to do? Preferably an assay that is simple and not too expensive as i have 30 extracts to test.
I always liked 3H-thymidine incorporation for proliferation assays. Then looking at DNA content by FACS to look for cel cycle arrest.
It seems then MTT assay should be able to distinguish btn cytotox and arrest. If signal goes below t=0, then there is tox. If signal stays at t=0 level, there is arrest. Of course, getting the t=0 data seems tricky.
good luck
Check these links, if you will:
http://search.vadlo.com/b/q?sn=158621799&a...icity&rel=0
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Hi Pronana,
i won't go for 3H-thymidine because it is radioactive with all the included problems: ceritification, waste disposal, .... 
We usually go for the WST-1 prolfieration assay - it is the most sensitive assay, best linear range and easier to handle than MTT or radioactivity
or Cytotoxicity Detection Kit plus (LDH) for cytotox assays.
You can do both assays in a 96 well plate which fits for your 30 samples (you can do them in triplicate on 1 plate). 
There is a good manual from RAS available:
http://www.roche-applied-science.com/sis/a...l_apoptosis.pdf
As far as I know the people from RAS offer the best range of Apoptosis, Cytotox and Cell Proliferation kits. They are a little more expensive, however they have the best kits and an excellent service including nice manuals and posters... should be worth to try. It`s not cheaper to try several cheap kits until finding the best... 
Buy cheap - buy twice!
Thanks all,
rosewater : I guess i'll have to scratch out the 3H-thymidine as my lab is not equip to handle radioactive materials. Refering to the MTT assay,What do you mean by "If signal goes below t=0, then there is tox. If signal stays at t=0 level, there is arrest."?? 
Senior_scientist : what is a WST-1 prolfieration assay?. I'm also tempted to use an LDH kit however my supervisor prefers more of the conventional methods (i.e : no kits). totally agree with your line "Buy cheap - buy twice!"
How about the neutral red assay for cytotox detection? I've seen some people using this assay for cytotoxixity.
however the principals of this assay is more or less similar to the the MTT i think. can someone shed some light ? Is this assay suitable to detect cytotoxicity?
[quote name='Pronana' date='Jul 16 2008, 04:49 AM' post='143881']
Senior_scientist : what is a WST-1 prolfieration assay?. I'm also tempted to use an LDH kit however my supervisor prefers more of the conventional methods (i.e : no kits). totally agree with your line "Buy cheap - buy twice!"
WST-1 is a tretrazolium salt assay (like MTT) but the product is water-soluble (WST - Water Soluble Tetrazolium salt; you do not have to dissolve with DMSO), it is more sensitive and has a better linear range...
Well, the kits are built on conventional methods, but are optimized and quality controlled for better reproducibility 
Maybe your supervisor just wants his students to be more busy in the lab 
Greez Chris
rosewater : I guess i'll have to scratch out the 3H-thymidine as my lab is not equip to handle radioactive materials. Refering to the MTT assay,What do you mean by "If signal goes below t=0, then there is tox. If signal stays at t=0 level, there is arrest."??
By t=0, I just mean the signal you get from the cells at Time=0. So the number of cells at the start of the experiment should remain the same, if cell proliferation is arrested...
It's not important.
There is also some reagent that measures the amount of ATP per cell that our HTS group used to use. It was more sensitive than MTT, but I don't have the info handy. Maybe it is what Sr scientist is recommending.
MTT measures cells with active mitochondria, so both cytotoxicity and proliferation, WST would be similar. You can probably buy WST without kit, but I wouldn't bother changing from MTT to WST.
I use BrdU incorporation for proliferation. You can get non-radioactive kits now, but you can also buy components if your boss is VERY opposed to kits. The overall cost may not be too different though. You can couple it to fluorescence or luminescence depending on what's available in terms of plate readers for you.
have you looked at the Promega website? they have a range of cyto-tox assays which are very good. i have used these instead of MTT. they have a range of different reagents depending on whether your looking at toxicity or proliferation - might be worth a look