Quantifying Immune Cell Populations

Peripheral blood cells were collected by retro-bulbar puncture from isoflurane anesthetized mice. Peritoneal lavage cells were collected as mentioned above. After red blood cell lysis, remaining cells were fixed with 10% methanol-free formalin for 10 min at 4°C and blocked with PBS/10% FCS for 10 min at 4°C. For flow cytometric analyses, cells were stained with antibodies as described recently [15 (link)]. Briefly, we used CD11b-Alexa Fluor647, SiglecF-PE, Gr-1-APC-Cy7 antibodies (BD Biosciences, San Jose, CA), CD115-PE-Cy7, CD19-eFluor605, CD3-eFluor450 antibodies (eBioscience Inc, San Diego, CA) for peripheral blood cells, and F4/80-eFluor450 (eBioscience), SiglecF-PE, Gr-1-PerCP-Cy5.5, CD19-PE-Cy7 and CD3-APC antibodies (BD Biosciences) for peritoneal cells. Intracellular neutral lipids were quantified by staining with BODIPY493/503 (1 μg/ml; Life Technologies, Carlsbad, CA) for 10 min at 4°C. Cells (1 × 10⁵ cells/measurement) were analyzed using an LSR II flow cytometer (BD Biosciences). Data were acquired using DIVA 6.1.2 software (BD Biosciences) and the analysis was performed using FlowJo (Treestar Inc., San Carlos, CA).