This is an ongoing problem in the field. People will deplete with Ly6G and attempt to confirm depletion by staining with Ly6G antibodies. This of course does not work because injected antibody is masking the flow-antibody. Like Nuruddeen, i've examined neutrophils with surrogate staining e.g. Ly6C mid/CD11b hi/ssc hi. And while good depletion occurs in the blood, many can remain in organs. I'd love to learn how to deplete neutrophils effectively. May be impossible considering the numbers generated daily.

I never checked the spleen, but in the peripheral blood they are depleted within 24 h and this depletion lasts more than 3 days. Daley at al described it really good: http://www.jleukbio.org/content/83/1/64.long

Like Anna kovtun, I never checked the spleen. I saw the nwutrophil depletion on 24 h, but 98% of neutrophil depletion I observed in 48 hours. There are different papers in different models that describe the anti-Ly6G antibody use.

Thanks Anna and Isabel. Isabel, can you tell me how much of the antibody you used? I'm guessing that it was the 1A8 clone right? I've checked after 18 hours and didn't see much change, even with 300 ug of antibody. I know the antibody bound to the cells though, as I tried to stain with the same antibody clone and the staining was gone. However, when I detected the neutrophils using CD11b and SSC-hi, they were still present.

Nuruddeen, I'm from Brazil, and here the availability of reagents is very scarce, because this we tried to use the smallest possible amount. We treated the animals with 50 ug/animal, maybe because this we saw in 48 h.

Nuruddeen, how did you inject antibody? Was it i.v. or i.p., or s.c.? Are you sure that you detected neutrophils, and not macrophages or NK in FACS? How were the values comparing to control?

This is an ongoing problem in the field. People will deplete with Ly6G and attempt to confirm depletion by staining with Ly6G antibodies. This of course does not work because injected antibody is masking the flow-antibody. Like Nuruddeen, i've examined neutrophils with surrogate staining e.g. Ly6C mid/CD11b hi/ssc hi. And while good depletion occurs in the blood, many can remain in organs. I'd love to learn how to deplete neutrophils effectively. May be impossible considering the numbers generated daily.

Agree with Anthony. Immunomagnetic separation with anti-Ly6G in vitro efficiently depletes neutrophils from suspension of splenocytes. However, we were unable to deplete neutrophils in vivo in the mouse spleen by injecting of 100 mkg of anti-Ly6G. Against, on the next and subsequent days after injection of mAbs they were accumulated in spleen. It may be, reasons for this are fast internalization of immune complexes from the surface of neutrophils, not allowing complement-mediated cytolysis or FcR mediated phagocytosis. Nevertheless, neutrophils coated by antibodies could be trapped by splenic FcR bearing cells. Second reason may be very active myelopoiesis which could be additionally stimulated in response to cell depletion. I should note that in mice extramedullary hematopoiesis can take place in the spleen. I think, another approach is necessary to deplete neutrophils. I would suggest cyclophosphamide (100-150 mg/kg), or sublethal irradiation. Although these agents are unspecific, they could be really helpful allowing to see decrese in neutrophil levels during ~1 week. As alternative approach, I would recommend to search for mouse strains, deficient in neutrophils: http://jaxmice.jax.org/list/ra1685.html

It may be, somebody could suggest anything better. I would be very interested.

Hello all, thanks again for your thoughts and suggestions. I ended up injected 500 ug of antibody IP and assessed neutrophil numbers after 24 hours. I was able to achieve a 75% reduction in the blood, which was sufficient to see an effect in the assay I was performing. I had to identify the neutrophils using alternative gating methods, but I'm certain they were neutrophils, as I was able to confirm my gating strategy in the isotype-treated group. I did notice that in some mice, the neutrophils were Ly6G low, but still detectable vs Ly6G negative, while in other mice, the neutrophils were completely Ly6G negative (due to binding of depleting Ly6G antibody).

24 pretreatment with neutrophil depleting antibodies can induce a number of protective genes. see Neutrophil depletion protects against murine acetaminophen hepatotoxicity: another perspective.

Hi all, I've now published my results in which I depleted neutrophils. Here's a link to the paper: http://www.ncbi.nlm.nih.gov/pubmed/25170619 . Thanks again for all your help. 

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What is the preferred root of Ly6G injection? IP or IV?

i.v. is far more effective, but you can do it i.p with enough antibody.

Has anyone assessed how much time passes, after a single injection, before the neutrophil population replenishes itself?

Agree with Antony. Those depleting antibody do not work. If you measure by flow cytometry, promotions might decrease, but if you assess their whole number, using basic blood smear analyser, the quantity is more than tripled per uL. We also assessed bone marrow changes after treatment, all precursors were largely increased upon treatment. Granulocyte depletion results in more granulocytes. 95% of published papers on the topic are bullish..

Rodrigo Guabiraba you have reference publication for the information you shared? If yes that will be greatly helpful. And which company antibody did you use?