Hello Charles,

To follow up Lisa's suggestion (thank you, Lisa), I'm a member of Tom Wynn's lab and would be happy to share our protocol to measure arginase activity and to answer any further questions. The overall procedure is described here and, from what you've described of your experiments, this approach ought to work nicely.

http://www.ncbi.nlm.nih.gov/pubmed/21462164

As alternatives, you might check Peter Murray's papers for Western blots of Arg1 and Arg2, and Stef Vogel has developed a method for staining Arg1 by flow cytometry. One suggestion of caution, though: Peter has screened antibody clones and found many work poorly, with both low sensitivity and low specificity for Arg1.

Please feel free to contact me directly by emailing [email protected].

Cheers,

Luke

Mouse? Human?

Hi Charly,

I've already been confronted to this kind of problem. Usually, to distinguish M1 from M2 macs, people are just checking the expression of marker genes by qPCR such as ARG1 (in mouse), RELMa, MR...for M2 and IL-6, iNOS...for M1. However, if you really want to show a functional activation of M2 through Arg1 activity, you can check NO production upon LPS activation and dose NOx compounds. If Arginases are activated, you should expect a decrease of NO production. Nevertheless, you will need to knock down Arg2 to prove that the effcet you observe is mediated by Arg1. I have tried collagen synthesis as well, but it is really painful, available kit are not good enough and you will get quite a lot of variability between experiment. Some people have set up hydroxy-proline production as well for that purpose (check Chris Scotton paper in UK for example), but this is not easy at all either. In any case, you will have to knock down Arg2. In macs, I was using a SMARTpool from Dharmacon and I was electroporating BMDM or RAW cells. It works very well.

Antibody against Arg1 on the market are pretty bad. The only one who works very well, I mean you can detect Arg1 basal expression in macs, is the one from Sid Morris in Pittsburg. However, on IL-4-activated macs, the one from Abcam is fine.

Hope it helps

Good luck in your experiments

Benoit

Thanks a lot Benoit, Pedro these are bone marrow derived macrophages taken from mice and cultured in teflon bags in M-CSF. They are lovely cells to work with! I'm trialing a drug that massively knocks down NO production in macrophages as determined by the Greiss assay, but I'd like to know if the resulting NO low cells are M2 or not.

Thomas Wynn has published several papers assaying for arginase production in the presence of Schistosoma. It is a plate based assay which may be amenable to your needs. As I recall, his papers are fairly consistent in their detail of this assay but he is also a very nice guy who would possibly assist via direct contact.

Hello Charles,

To follow up Lisa's suggestion (thank you, Lisa), I'm a member of Tom Wynn's lab and would be happy to share our protocol to measure arginase activity and to answer any further questions. The overall procedure is described here and, from what you've described of your experiments, this approach ought to work nicely.

http://www.ncbi.nlm.nih.gov/pubmed/21462164

As alternatives, you might check Peter Murray's papers for Western blots of Arg1 and Arg2, and Stef Vogel has developed a method for staining Arg1 by flow cytometry. One suggestion of caution, though: Peter has screened antibody clones and found many work poorly, with both low sensitivity and low specificity for Arg1.

Please feel free to contact me directly by emailing [email protected].

Cheers,

Luke

Thanks so much, that's really helpful. I'll see if I can get the reagents together to have a go if the qPCR suggests that there is increased transcription of Arg-1.

What a great discussion. Charly, I'm just curious regarding your extraction of macrophages from mouse bone marrow, as to whether you select out cells such as regulatory T cells...in other words, are you sure the only cells you are working with are macrophages? Also, do you culture your cells in ambient oxygen (21%), or a lower concentration? As you can tell I'm trying to help by figuring out a few of the variables that may confound studies of cells once they have left the in vivo environment for the in vitro environment...

Best wishes,

Chad

Hi Chad,

I'm using the protocol developed by the opthalmology group here. I extract the cells by flushing the bone marrow out with DMEM then we culture them for 8 days in high glucose DMEM supplemented with FCS, horse serum, M-CSF, L-glut and pen/strep in teflon coated bags at 37 degrees in 5% CO2. We don't select out cells as the culture conditions don't support T cell growth as they remain unstimulated. Also, correct me if I'm wrong, there shouldn't be many T cells in the bone marrow anyway? We have found that the macrophages derived from this protocol are over 95% pure: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397322/

Best wishes,

Charly

Simple method.But it will not differentiate between arginase 1 and 2.

Br J Pharmacol. 2012 Jan;165(2):506-19. doi: 10.1111/j.1476-5381.2011.01584.x.

Hi Charly, lately I was also planning to detect Arginase-1 as one of the hallmarks of M2, which troubled me a lot. Papers I found out mainly applied the protocal you mentioned above, if that, I have to gather so many reagents. Of course, qPCR is also an alternative, but I am not familiar with that (sigh). Thus I really want to know if you find something good to work it out. THX so much!!!

Re FACS I would combine cell surface markers with Arg-1 (intracellular). We used an Ab from R&D systems that worked fairly well. It should be possible to measure both NOS and Arg activity in lysates and their activities should be inversely proportional as these two enzymes share the same substrate. I would prefer western blot to qPCR as a readout but am not aware if there are good Abs which distinguish between Arg-1 and Arg-2.

When I elucidated M1 and M2 macrophages, I was fortunate to have an amino acid analyzer in my lab. So, I could measure the Functional Product of arginase, Ornithine. So, if cultured cells, I would collect supes and find an aa analyzer. Or, a company that does it commercially. You'll save yourself a lot of trouble. Measuring the signature M1 product (NO) is a very easy colormetric assay as described by others. It is nice to see more and more wanting to measure the signature M2 product arginase because some have gone off and measured a variety of other cell markers, or other products, that are not repair - related as Ornithine. And, become confused about "subset". Paper that may help attached. PubMed Sidney M. Morris for arginase, and non - amino acid analyzer assays for Ornithine. He's a key expert in that area.

Here's original M1/M2 Paper if also helpful. Good luck.

Out of interest, is NO considered a M1 product moreso than TNFalpha? The NO assay is easy. Measuring ornithine seems a good idea.

If I am not mistaken, due to its cytoplasmic colocalization with ornithine decarboxylase, arginase-1 would preferentially induce the synthesis of polyamines from ornithine to lead cell proliferation and growth whereas due to its mitochondrial colocalization with ornithine aminotransferase, arginase-2 activity would lead to the synthesis of proline, the precursor of collagen and glutamate (Li et al., 2004). So perhaps AA measurement would be a way to distinguish between the activity of the two isoforms?..

When we were looking at competition of arginase and NOS for arginine (in foam cell macrophages produced in vivo), I measured RNA (qPCR, SSH) and protein (Griess assay for NOS activity, urea assay for arginase activity and HPLC for ornithine and proline (ie aa) production) levels. These days you can't just measure something one way - has to be several different ways!

We ended up thinking of NOS high cells as more M1 and Arg-1 high cells as more M2a. I should think that Arg-2 high cells are M2C, but the HPLC results were inconclusive.