Dear Rana

SGC-7901 has not DPP receptor? 

if SGC-7901 has no DDP receptors, then it shouldn't even respond to the low concentration of DDP, because the concentration of the second drug is constant in all the groups 10 microMolar. 

Hi Rana - that is puzzling. By "DDP" are you referring to Cisplatin?

The most obvious answer is that you are getting a false positive result.

The CCK-8 kit works by measurement of formazin production - if a reducing agent is present then this will generate formazin (from the Wst) and give a false background.

The first thing to check, therefore, is what colorimetric reading you get with increasing doses of DDP in wells that contain no cells (negative control at all concentrations). Include the plant extract in case there is a weird interaction going on that is generating a reducing agent.

If in the absence of cells you do not get an increase in colour then something else s going on. You will then need to repeat the experiment but instead of using a CCK8 kit seed the cells equally (in triplicate per condition) and incubate for several days. If cell proliferation really is going up then at the end of i.e. 72 hours there will be more cells in the wells with the higher dose. Fix, stain with i.e. dapi and physically count the cells.

Hope this helps,

Gar

@Gary.. DDP means Cisplatin, all the false positive conditions have been ruled out. Even under a microscope, we can see dead cells as compared with higher drug concentrations. I have already tried cell counting as well. Actually, the color is the true representative of the number of cells in those particular wells.

That is tricky to explain then. All I can think of is that the effect you are seeing is being caused by a heterogeneous cell population.

What passage level/cPD is your cell line at?

The only explanation I can think of is due to a complicated series of events and is thus not likely to be the case.

i.e. during prolonged passaging in vitro a sub-population that is DDP sensitive has begun to emerge (sDDP) and, as it is faster growing than the DDP resistant cells (rDDP),  it has come to dominate the population overtime. Furthermore these sDDP cells exert an inhibitory effect on the non-sensitive (somehow - exosomes, paracrine or what ever effect). At the lower drug concentration some of the sDDP cells begin to die- but not fast enough to release growth inhibition of the rDDP - so a drop in the growth of the culture overall. At higher concentrations the sDDP die off quicker, inhibition is removed and the rDDP grow faster.

As I said  - an unlikely situation. However you might want to try a few things.

The easiest is to go back to earlier cell stocks (or buy some new cells), grow up and retry with these cells to see if the effect persists.

The second thing may be to maintain the cells in culture in the presence of the lower level of  DDP (no extract) and see if over time these cells recover proliferation as a rDDP population of cells emerge.

The third thing to try would be to obtain another rDDP line and run alongside in parallel (from an early passage batch). If the same thing occurs in this line then it is likely that some other factor (wrongly made solution/poltergeist/gypsy curse) is at play.

Finally - and I don't recommend this merely raise it as a possibility - you could generate some clonal populations (via i.e. limiting dilution), grow up various sub-clones and check their resistance to DDP. If sub-populations are present you may get variable response to DDP.

Sorry I can't be more help but if you ever do find out what is causing this please post!

Gary

Thank you, Gary, appreciate your suggestions and time, i think I will try to generate some clonal population. 

OK good luck. Limiting dilution is time consuming and be aware some cells are sensitive to low density. If they don't grow try using conditioned media (media from cells at ~ 60-80% confluence in flasks). Sometimes this supplies necessary paracrine factors.