to find out role of Neutrophils and machrophages in lung inflammation after drug treatment isolated mice lung single cells and by the help of Percoll separate leukocytes..than stained with GR-1 and F4/80 separately because both are FITC labbeled for flowcytometry. But now confused which cells i have to select for Neutrophils and Machrophages stained with Gr-1 or F4/80. I donot understand how i gate or which cell collection represent neutrophils. please see attached file.either R2 or R1 represent neutrophils?or i should have to gate differently.
Hi Norman - we do a lot of lung flow, for starters, how many colours is your flow? I'm not sure what benefit Percoll will have for you, as it's easy to tell the difference between macrophages and lymphocytes and non-immune cells just by adding in a CD45 antibody to your cocktail. Many cells are lost during Percoll so if you're about to do flow with counting beads, your counts will be way out. If, however you're extacting cells for culture then I get it!
Neutrophils will be CD45+, CD11b+ and Gr1+ (Gr1 is a shared epitope between Ly6C and Ly6G) but they are F4/80 negative. I use Ly6C and Ly6G separately as it allows you to also distinguish between Ly6C high and low monocytes.
Macrohpages are tricky but alveolar macrophages in their resting state are CD45+ CD11b-, F4/80 positive and CD11c positive. Macrophages also autoflouresce in the FITC channel so using F4/80 in there is a good move as it helps distinguish them from dendritic cells. There are other markers you can add in such as MHCII or Siglec F, depending on how many channels you have. After an insult lots of CD11b+ cells appear.
As for your graphs - I can't help as you can't separate neutrophils just on FSC and SSC. You need some markers in there.
I usually run whole lung, then gate first on counting beads, second on CD45 and take the CD45+ cells. I then gate those on CD11c and F4/80 - the macrophages are the top right population. I then gate the rest on CD11b and take the CD11b+ cells and gate those on Ly6c and Ly6g - which will give you a clear neutrophil population in the top middle, and two monocyte populations underneath.
If we had more colours I'd be able to add better macrophage markers in but as preliminary work this is the best I can do!
Charly provided a solid response. A couple of clarifications to your protocol might be helpful. First how are you obtaining single cells. Presumably, collogenase/DN'ase but is this correct. Always be careful that you have no contaminating proteases in your enzyme mix as they will strip membrane markers. Second, what Percoll gradient are you using? Is it continuous or discontinuous? Which bands are you calling the myeloid cells? Having said this the lymphocytes, macrophages and granulocytes would be at different densities in a Percoll gradient. Please see papers by myself and Bob Wiltrout some time ago on a 5 step gradient. Having said this, a murine Ficoll Hypaque gradient will largely separate parenchymal from non- parenchymal cells. Alternatively, as suggested doing the flow without gradient separation might also prove useful. Lastly, consider removing doubles and apoptotic cells from analysis.
Depending on the drug used to treat the mice you might want to also consider the presence of MDSCs and myeloid progenitors in the lungs (add Sca-1 and Lin markers to panel). When working with myeloid cells antibodies to CD115 and CD66b can also be useful. Similar to what Charly is suggesting when doing studies of this nature we use at least 3 lasers and 8-10 colors.
Thank you Carly and Sir James for detailed and quick reply. Sir James Your expert advices always helpful for me. Now i am studying all the points you mentioned and will design experiment . Thank you once again
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