Activation may be due to intracellular contamination by mycoplasma (hard to see), virus or bacteria (easy to see). or by droplet of other culture cell line if several cell line manipulation Under the same Hood or work session.

Also be sure you re using a fresh aliquot and not more than 6 - 10 passages of your Raw cells. Avoid to  detach your cells by scraping after EDTA treatment, preferably use cold EDTA solution.  and be sure your watter is purified.

In addition to a microbial contamination there is also the possibility that your glass/plastic ware, media and water may contain lipopolysaccharide, which could activate your cells. Care should be taken to remove LPS from all utilities, media and water - if you are not already doing that.

Best regards

Lasse 

First, I'm unclear as to what you mean by activated?  Are you testing for TNF production or are you seeing a morphological change in the cells?

Either way, in addition to ruling out bacterial contamination (as the others have suggested), I suggest you passage your cells by scraping and not by chelating.  If you insist on using EDTA, I recommend removing the majority of EDTA from the cell suspension before you subculture.  It is very easy to deplete the free Ca2+ and Mg2+ from the culture media if there is excess EDTA around.

Finally, another source of LPS contamination is the autoclave.  If you are using autoclaved materials, you might want to try using certified non-pyrogenic versions of them. 

What do you mean by activated? RAW 264.7 look spread and "activated" as a primary macrophage might, but is indeed not activated until chemically promoted to.

Also, RAW 264.7 are meant to be passaged by scraping, not chemical dissociation.

Dear Ho,

Very good question! I suppose that you, just like me, are observing an altered morphological phenotype indicating and "activation" (although it might not be activated in the sense of protein production). We have been using these cells for quite a while and experienced this phenotype in the beginning. This is what I was recommended and which works for us: 

First, which was already mentioned, these cells appear to be very sensitive for high passages, so you should use fresh aliquots.

Second, also mentioned, the cells are not very sticky which is why EDTA or Trypsin is not necessary. Simple pipetting is sufficient in my case.

Third, I was recommended by our provider to use 1% HEPES, which really seems to make the cells happier and maintain their circular morphology. Change medium frequently although the cells are not that metabolically active that the medium change color that fast.

Lastly, as indicated, make sure that your cells are not contaminated with mycoplasma for example.

Good luck with your experiments!

I suggest you to detach the cells with Basic DMEM, but not with PBS. Then digest whe trypsin contained with EDTA quickly. Like Partrik said—— these cells appear to be very sensitive for high passages, so you should use fresh aliquots.  When we found the cell morphological phenotype changed,  we recovered the fresh cells from liquid nitrogen. Maybe this would help you.

Another tip, add the 1/200 Ciprofloxacin  would work better. 

Like others have said above, I suggest:

1. Checking the endotoxin levels in your media and PBS. Are you making your own or purchasing? When we made our own, we purchased endotoxin free water.

2. Using the RAW264.7 cells at low passage numbers. We saw significant changes after about passage 20 so we would restart with a fresh vial.

I am using the same cell line, Heat inactivated FBS would make a big difference. As well, the passage number played a role.