does MG132 dissolve in culture media? pls im desperate for help

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cyrine
cyrine's picture
does MG132 dissolve in culture media? pls im desperate for help

I have been treating a cell line with increasin concentrations of MG132 10-100uM).i make a 1mM stock solution in DMSO then dilute it down in media. however i am getting very variabe results each time. some times a falt response of all concentrations giving 40% cell death and other times ups and downs in the curvi dont understand why. i tried many times. i am using an LDH assay and an ATP assay

samm
samm's picture
What cells are you using?

What cells are you using?
Do you use them at one consistent level (e.g. 70% confluency, 5% serum, 2-5 passage etc). Since MG132 is a very good proteasomal inhibitor - and as such is blocking a critical cell pathway (most proteasome component knockouts, and all functional ones, are lethal), you need to ensure that all other conditions are uniform.

samm
samm's picture
Also, try titering across a

Also, try titering across a wider range - some cell types are more sensitive than others. If there is no published literature for your cell type, I'd recommend a pilot assay with a 1000x range so that a sigmoid profile is seen.

cyrine
cyrine's picture
thank you for your answers

thank you for your answers

yes i am using consistent cell confluence and other parameters. the problem is an increase in the concentration of MG132 within the same experiment is not giving me an increase of cell death. i have tried very low concentrations and very hig. in fact ive tried from 0.1 uM to 100 uM

Guy Sovak
Guy Sovak's picture
I have used MG132 at 10uM at

I have used MG132 at 10uM at a dilution of 0.1%DMSO and I got good results with nearly no cell death.
The treatment was done on BHK and HeLa cells for 3Hr.
What cells are u using?
Guy

annafreud52
annafreud52's picture
 Hi, I am having the  same

 Hi, I am having the  same trouble as cyrine's. My cell line is SH-SY5Y neuroblastoma one. My problems are exactly the same method, the same concentration range, the same dilution method, the same results and the same assay. I am trying to find EC50 concentraion of MG132 that kills 50% of cells and papers say it should be around 2- 20uM. Can any one help? 

Chin Fen Teo
Chin Fen Teo's picture
Hi Annafreud,

Hi Annafreud,

It isn't clear to me the problem that you are encountering is either the irreproducible results or the cells are dying (or not dying). Can you please elaborate?

annafreud52
annafreud52's picture
 Hi Pippuri, thanks, to be

 Hi Pippuri, thanks, to be more specific, the cells are not dying (ie below 30% at even 10uM and 100uM) as assessed by LDH assay. 

Chin Fen Teo
Chin Fen Teo's picture
Hi Annafreud,

Hi Annafreud,

There are couple possibilities-

How did you prepare MG132? While MG132 is very stable in organic solvent, it is rather unstable in aqueous condition. It has to be prepare fresh in the aqueous solution (ie. media) on the same day of experiment.

The cells that you are treating MG132, what is the passage #? Often time, when the cells have been passed for too many times, they will be resistant to many treatments!

One other way to evaluate whether MG132 is working properly is to perform a western for Ubiquitination, since accumaltion of ubiquitinylated proteins is one of the direct outcome of MG132.

Good luck.

annafreud52
annafreud52's picture
For some reason my ID,

For some reason my ID, annafreud, was not working so I have created a new ID, annafreud52. 

Could anyone share any experience on what his/her EC50 of MG132 was around? 
Vilotti's paper below says 1 and 5uM MG132 gave over 80% of cell death in SH-SY5Y cells after 16 hr as assessed by MTT assay. 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278732/