Fluoresbrite green fluorescent yg plain microspheres

Monocyte recruitment to aortic root atherosclerotic lesions was evaluated following atherogenic diet feeding for 8 weeks using previously described methods^{64 (link)}. Monocytes were labeled in vivo by retro-orbital injection with 250ul of 1 μM Fluoresbrite green fluorescent (YG) plain microspheres (Polysciences Inc., 17154) diluted 1:4 in sterile PBS. Blood was obtained 24 h later to perform flow cytometry to assess YG beads incorporation efficiency. Briefly, monocytes were identified by staining for CD45 (CD45: Phycoerythrin (PE)-Cy7, BD, 561868, Clone 30-F11, 1:100) and CD115 (CD115-PE, BD, 566839, Clone AFS98, 1:100), and characterized to be Ly6C^lo or Ly6C^hi (Ly6C-APC, BD, 560595, Clone AL-21, 1:100) by BD FACSCalibur™ Flow Cytometer. Green fluorescence revealed YG-bead abundance. The incorporation of YG bead-containing monocytes in aortic root sections was determined by visualizing green fluorescence 24 h following intravenous YG-bead injection. YG-beads per aortic root section were counted and the quantitation was corrected for labeling efficiency in circulating monocytes.

At week 4 after initiation of the atherogenic diet, circulating monocytes were labeled by intravenous injection (100 µl per mouse) of 1 µm Fluoresbrite green fluorescent (YG) plain microspheres (Polysciences), 3 days after clodronate liposome (Liposoma) injection. Eight weeks after initiation of high-fat diet feeding, mice were anesthesized, and an antibody that binds CD11b (clone M1/70, 1 µg, PE) was administered intravenously. The carotid artery was exposed, and the interaction of myeloid cells with the carotid artery was recorded using a ×10 water-dipping lens.