Co-incubation of LPS and drug - (Apr/03/2015 )

Hi!

I'm working with J774 macrophages. For my experiment, I have to induce inflammation in these cells by adding 0,1µg/ml LPS. At the same time I have to co-incubate my test drugs (100µM, 10 µM and 1 µM) with the medium containing LPS to see if they inhibit some inflammatory pathways. I make a batch of this 0,1µg/ml LPS in medium and then aliquot for every test concentration per well.

My problem is that I don't know how to keep the LPS concentration the same in every well while also adding the drugs...

Every well in my 12-well plate is filled with 1mL with 0,1µg/ml LPS, but if I need a concentration of e.g. 100µM test product I need to add 100µL of a 100mM stock of my drug to 900µL medium with LPS to get to the final volume of 1mL. But by doing this I dilute the LPS, so it's not the same in the 100µM, 10 µM and 1 µM concentrations.

How can I do this correctly, or is this indeed the best way?

There are a couple of options here - either make the drug stock at such a concentration that adding a small volume (perhaps 1-10 ul) would give you your 100 uM (you can dilute for the others, so that you are adding the same volume to each well of different concentration).

You could also compensate for the added volume with your LPS dilution (e.g. make 0.11 ug/ml) then add 100 ul to each well of your various drug concentrations drug (you would need to compensate the drug amounts added too).