Serum from LPS-injected mice contains NPA. (A) NPA-mediated generation of granulocytes clusters in vitro from bone marrow cells of G-CSF^−/−/GM-CSF^−/− exposed to medium conditioned by LPS-challenged G-CSF^−/−/GM-CSF^−/− MEFs (May-Grünwald/Giemsa stain, original magnification ×40). Inset: same field (original magnification ×60). Micrographs were acquired on cytospin slides stained with May-Grünwald-Giemsa on a Nikon 90i microscope (Nikon, Tokyo, Japan) fitted with a 40×/0.6 numeric aperture Plan Fluor dry objective or with a 60×/1.4 numeric aperture Plan Apo oil objective, a DXM 1200c camera (Nikon), and NIS-AR image acquisition software (v 2.30; Nikon). (B) NPA in sera from G-CSF^−/−/GM-CSF^−/− mice injected intraperitoneally with PBS (○) or with LPS (30 μg/mouse, ■). Sera from 3 mice per experimental point were individually tested for NPA as described in “NPA assay.” Data are mean plus or minus SE for each group. One unit of NPA is defined as the activity required to generate one granulocyte cluster from 5 × 10³ bone marrow cells after 4 days in a liquid culture assay. Error bars represent SE.