MDA-kb2 cells - problem with luciferase expression - (May/18/2015 )

Dear all,

I am working with MDA-kb2 cells which are supposed to be used for agonist/antagonist screening of androgen receptor. They contain luciferase gen. After weeks and months of optimizaton, comparing of protocols from differrent researchears I've never obtained a response - which is measured by luminescence of luciferase. One of the problem I suspected was contamination of mycoplasma...but after 3 tests of different cultures I confirmed that  cells are mycoplasma-free. Cells in general grow, and are viable. However after some passages it seems to grows worse (according to literature this number of passages is still not as much as the one that is reported to decrease the viability of cells).

I am not well aquinted with this cells neither with cells in general Could you take a look of some pictures of them? Maybe there are symptoms that ringing a bell and can be helpful to solve this problems.

link to some pics: https://onedrive.live.com/redir?resid=73D9227DA7F392C9%2111022

more about this cell line: https://www.lgcstandards-atcc.org/products/all/CRL-2713.aspx?geo_country=es&slp=1#generalinformation

thanks!

Andrzej

I'm not familiar with this line, but the cells look OK. There might be a higher number of floating/rounded cells in the population than one might expect, but this may be a natural part of the cell behaviour.

Do you have them under selection?

Are your luciferase reagents fresh?

Thanks very much for answer! :)

Actually I don't have them under selection, believing as It's commercial line they should be ok, but probably that's a good way to make sure there is no problem with genes expression. I will try next time culture with geneticin. We will see..

Regarding luciferase kit I think it's ok - I used a new one and everytime doing measurement a luciferase standards were checked parallel and I obtained signal from pure protein.I also try spiking my medium with luciferase and signal is ok, so there is nothing in the mediom that could cause an inhibition of enzymatic reaction.

Are you using the L15 medium the ATCC recommend?

Did you check out the references on the characteristics page from the ATCC? They might have more information in those publications.

I use L-15 from Life Technologies as it's much cheaper than the one from ATCC. I studied the composition of medium compound by compound and it is actually the same. Just the concentration of L-Lysine (0,075 g/L vs. 0,09 g/L) and L-Tyrosine (0,3 g/L vs. 0,43 g/L) is a bit smaller in that from life tech.

Finally I ordered new cells, so as to make sure that nothing was spoiled in previous steps. They were growing very well and yesterday I made first subculture. I've seen a kind of weird outgrowths (?), don't know even what's called. Anyone knows what is that? I marked some of them in the picture.

*I divided subculture and some of the flasks have antibiotics, some no. All look the same.

Those look like blebs from apoptotic cells