The Griess assay protocol says that the equal volume of supernatant and Griess reagent is to be mixed and incubated. Proceed with your protocol (steps 1 to 4). Whatever the volume of media you uses, just don't change it.

Answer to your query: Consider that you have seeded the cells and once it has grown, you are ready for treatment. Now when you will do the treatment, anyway way you will use fresh media for the treatment set (i.e with your compound). Now, at this moment, you can give media change to the control set also. Keep media volume equal of media for both sets. Now, after completion of treatment, you can take an equal volume of supernatant from control and treatment conditions and proceed with the Griess assay.

If you can manage with 100ul of media it could be beneficial, more media sometimes dilutes the NO production and reading comes low.

Saurabh Mandal Just to be clear, base on what you are saying, you will always replace the overnight media with fresh media + compound. You will not just directly add the treatment compound into the media after 24 hr of incubation?

Hi,

Totally agree with previous commentaries.

In general, it would be better to remove old media together with any debris to prevent any unexpected effects. Practically it is very useful to have a small volume of old/fresh media (f.e. 50 ul per well in 96wp) before the addition of the fresh media with/without LPS.

At the same time in certain situations, the changing of media could be an excessive step. I was working with peritoneal macrophages harvested with HBSS with Ca Mg and later added fresh DMEM/F12 10% FBS with or without LPS. This approach works pretty well. Just be sure you included cell-free wells in your template to have appropriate negative control for the basal level of nitrite. The keeping of the original lavage liquid was important in our setup to prevent Mf activation due to changes in media composition (surfactant and components of peritoneal liquid) and elimination of other cells present in the peritoneal cavity.

If I will perform experiment as you dealing with I will ask myself:

How the fresh media could affect NF-kB activation pathway?

What is the nature of effect casing by tested compound?

Could mentioned compound be a scavenger for ROS/RNS or it somehow affects signaling?

What other questions could ask reviewers related to the used model?

If we are talking about dividing cell population and experiments dealing with the evaluation of gene expression in addition to NOx production I would think about overnight starvation (media without glutamine and FBS) of the cells to synchronize them and to have a more uniform response. From my previous experience there are a lot of uncertainties raised in the experiments on deviding cells.

Hope you will find useful my comments.

Have a nice experiment!