http://cvi.asm.org/content/early/2012/04/30/CVI.05715-11.full.pdf

Thank you very much for your answer.

I treated my rats with CdCl2 and I have successfully isolated mononuclear subpopulation from digested lung tissue but I had a lot of trouble with granulocytes. Histopathological evaluation of H&E stained lung tissue slices showed increased number of granulocytes in treated group compared to control. I have done MPO assay and results implied their increased number as well. I used my protocol for mononuclear/granulocyte subset isolation from peripheral blood. If I ever get granulocytes from lungs I would like to examine cytokine production/gene expression, expression of functional markers, proliferation...

I will try with your protocol. Thanks a lot again

Hi Marina,

Here is an excellent reliable method for the isolation of total lung leukocytes, which is almost certainly your first step (see Hardy et al, J Immunol 2012, 188(3):1431). It contains the granulocyte fraction.

The right ventricle was perfused with 5 ml Ca2+/Mg2+- free HBSS (no. 14175095; Invitrogen) with 0.01 M EDTA, pH 7.2. Lung and draining LN were chopped with a tissue chopper (Mickle Laboratory Engineering Co. Ltd, Gomshall, U.K.). Tissue fragments were digested in collagenase type III (1 mg/ml; Worthington, Lakewood, NJ) and DNase type I (0.025 mg/ml; no. 1284932; Roche Diagnostics, Sydney, Australia) in a volume of 7 ml at 25°C by manual pipetting for 20 min. The reaction was stopped by adding one 10th volume of 0.01 M EDTA and mixing for 5 min. The cell suspension was filtered through a 70-mm cell strainer (BD Falcon) and underlaid with 1 ml 0.01 M EDTA in FCS prior to centrifugation (350 3 g, 4°C). The cell pellet was resuspended in red cell lysis solution for 3–5 min (no. R7757; Sigma-Aldrich), diluted to 10 ml in RPMI 1640 and 10% FCS, and underlaid with 1 ml 0.01 M EDTA in FCS prior to centrifugation (350 3 g, 4°C). Cells were resuspended in staining buffer [3% FCS, 3% pooled normal mouse serum, 5 mM EDTA (pH 7.2), and 0.1% Na-azide in Ca2+/Mg2+-free HBSS], and viable cells counted in a hemocytometer.

Following this you could perform a density gradient separations using Nycodenz or Percoll. My own experience with trying to purify DC populations by density gradient hasn't been overly successful (ie trying many different densities etc). However, if you want really pure granylocyte populations you could sort the cells by FACS, gating on neutrophils as CD11bhi Gr-1hi Ly6G+ (see Daley et al., 2008, J Leukoc Biol 83:64).

Good luck,

Charles

You could use this panel for granulocytes: CD11b+LY6G+LY6ClowF4/80–CD11c. I don t think it is good idea to use GR1 and Ly6g, Gr1 recognizes both LY6C and LY6G.

Find below a very useful paper:

http://www.nature.com/nri/journal/v12/n4/pdf/nri3175.pdf

Good luck

Hi all,

Dominic, yes, I think you would be right just using CD11b and Ly6G (I was looking at a plot in the paper by Daley et al.).

Marina, there is some really nice phenotyping in the paper by Plantinga et al., 2013 (Immunity 38:322), where the addition of Siglec-F and CD11c (and possibly also MHCII, as the neutrophils should be MHCII neg) would allow discrimination of eosinophils as well, another important granylocytic population in the lung!

Cheers,

Charles

Thank you all for good ideas. I will try with modified protocol for lung tissue digestion and than I hope that some of Percoll gradients will do the separation correctly. It would be easy to separate granulocytes using FACS, but my cell sorter is not working and my analysis are limited because I have only a few antibodies available for work.