To induce a good inflammatory response in RAW cells, make sure your cells are cultured and maintained in such a way that the cells are more or less in their pristine (unprimed, undifferentiated) state. Under the microscope , such cells should be roundish and bulging, with very limited arborization (protrusions or dendrites) . In regular maintenance (passaging), we use high-glucose DMEM, supplemented with 10% HI-FBS (heat inactivated) and glutamine/glutamax. For culture before and duration drug treatment, glutamine or pyruvate will dampen the inflammatory response (see documentations in literature), and so you may consider taking them out in the medium if you want a robust response. For LPS, prepare it at 5-10 mg/mL a s stock solution (usually endotoxin-free water), aliquot that in small sizes (30 ul per tube for example), and freeze at -80C or -20C until use. Avoid repeated free-thraw of the LPS stocks. Once thawed, keep that at 4C and use it up soon as possible. We found that 100-200 ng/mL LPS (Salmonella, or E. coli K12) is sufficient for inducing RAW cell activation. You can alternatively challenge the cells with LPS alone, LPS plus IFN-gamma (mouse/human), or IFN-gamma alone (50 ng/mL). The seeding density for RAW in 96-well assays is typically 1-2 x 10^5/mL, good for 1 day observation.

For references, we have described how to get RAW cells working in inflammation models in imaging and microplates in the following papers (See SI): (1) Article HKOCl-3: A fluorescent hypochlorous acid probe for live-cell...

Article Fluorescent Probe HKSOX-1 for Imaging and Detection of Endog...

Dear Dr.Wong,

Thanks for the detailed response, certainly it is very useful for us in standardization of the experiment..

If you are having to use 10ug/ml LPS, something is wrong. 10-1000ng/ml should be able to evoke an inflammatory response. perhaps try a different batch of LPS.