Cell Proliferation assay

[Idioteque]

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So, I need to measure toxicity of a nucleoside. What is the best assay that you guys have used to measure cell proliferation? I'm thinking of using one that measures MTS (or MTT) bioreduction into formazan (Promega). Does anyone have experience with this and can recommend a better approach? Thanks in advance. 👍

 

[xanthines]

Will trypan blue give you what you want? I'm not too familiary with toxicity/toxicology stuff, but the trypan blue exclusion dealio will roughly tell you how many cells are dead vs alive. Sorry I couldn't give more help.

-X

So, I need to measure toxicity of a nucleoside. What is the best assay that you guys have used to measure cell proliferation? I'm thinking of using one that measures MTS (or MTT) bioreduction into formazan (Promega). Does anyone have experience with this and can recommend a better approach? Thanks in advance. 👍

 

[Idioteque]

yea, tryptan blue is the method I've been using but I want a more accurate assay (I keep counting dead cells).

 

[Ol'DocToxic]

So, I need to measure toxicity of a nucleoside. What is the best assay that you guys have used to measure cell proliferation? I'm thinking of using one that measures MTS (or MTT) bioreduction into formazan (Promega). Does anyone have experience with this and can recommend a better approach? Thanks in advance. 👍

MTT is fine for cell tox studies, but technically speaking it's a cell viability assay, not a cell proliferation assay. If you don't want to solubilize with DMSO as in MTT and you want a better indicator of cell number, you can use an assay that employs the protein-binding dye Sulforhodamine-B (SRB). SRB is water soluble and thus the assay is a little less messy. It measures the amount of cellular protein in the cell culture, which is a pretty good indication of cell number. However, it will consider the protein on a dead cell equivalent to the protein on a live cell. Of course, the only true measure of cell proliferation is counting actual live cells, so trypan blue is the horse's mouth so to speak.

One alternate method you could investigate using is called a clonogenic assay. If your cells are anchorage independent, you can make a suspension of single cells and plate them into a semisolid medium (like low % agarose). The cells divide and form colonies that you can stain with MTT and count under a microscope. This assay would be specific for proliferation.

PM me if you want more details.

 

[Doctor&Geek]

Look up brdu incorporation, and cell counting using a Coulter counter.

 

[Treg]

To measure proliferation, you can use 3-H thymidine incorporation. If you are less excited about using radioactivity, you can use Brdu (as mentioned) or CFSE with a flow cytometer.

🙂 Treg

 

[Vader]

I regularly use an anti-phosphohistone H3 antibody (upstate).

 

[byong_soo]

Couldn't u just count the live cells with tryptan blue. One of grads in my lab is also working with MTT, and I am quite sure that she just uses trypan blue. Isn't it easy to tell from live from dead ones?

 

[logos]

We've done some work with nucleoside analogue toxicity in the past, however weve used enzymology methods primarily to assess the toxicity. We just compare how the analogue differs from the nucleotide it emulates in terms of kinetic constants with purified polymerases.

In culture the first thing I would think of would be just to synthesize the triturated nucleoside (or alpha 32P nucleotide?). That would let you measure the amount of incorperation and get some rudimentary kinetics in cell culture. Another option for measuring the incorperation in cell culture would be spectroscopy. We used to work with a fluoronated nucleoside, so what we could do (in theory, never did it) is just send purified DNA of known concentration for that elemental analysis spectroscopy thing. They basically just burn it and determine the amount of each type of atom (eg the amount of F = amount of incorperation).

I know those arent in vivo assays like you wanted, but maybe it'll be of some use...

Best of luck!

 

[PBRmeASAP.net]

tritiated thymidine or BrdU... simple, proven, accepted worldwide...

 

[JayQuah]

I've used an MTS assay and it didn't work that well.

 

[Ol'DocToxic]

I would be careful about using 3H or BrdU incorporation assays. You said you wanted to measure cell proliferation. These assays measure DNA replication and repair. If your nucleoside damages DNA, causing the cells to crank up their DNA repair activity, then your incorporation assay results may be misleading with respect to cellular proliferation. I know this is kind of splitting hairs, but, believe me, there will be some anal-retentive reviewer out there that will ding you on this point and you will go insane because you have to repeat a ton of cytotox studies to assuage the outlandish concerns of one nit-picking scientist.

 

[Time]

you got to be more specific, but if it is just looking for dead vs. alive cells, you may want to try Live Dead Assay(you may wanna pm me if you dont find anything on it, i think it is either sigma, or promega).....

it labels nuclei of dead cells red, while all cells that are alive and doing well will only label green. cells that are dead will have a green halo, too.

Very easy to use.

hope this helps.

So, I need to measure toxicity of a nucleoside. What is the best assay that you guys have used to measure cell proliferation? I'm thinking of using one that measures MTS (or MTT) bioreduction into formazan (Promega). Does anyone have experience with this and can recommend a better approach? Thanks in advance. 👍