F4 80 efluor450

In vivo macrophage migration into epididymal fat pads of mice was performed as published earlier 4, 23. Ten‐week‐old male C57BL/6 wild‐type (WT) mice purchased from Charles River Laboratory (Wilmington, DE, USA) were used. All mice were fed with standard chow and water ad libitum and experiments were approved by Yale University's Institutional Animal Care and Use Committee (2015‐10756). Five mg/kg LPS (Sigma‐Aldrich, St. Louis, MO, USA) only, LPS together with either 20 μg mouse D‐DT or the equal amount of mouse MIF was injected into the epididymal fat pads via a small incision to induce local adipose tissue inflammation. Control mice were treated with the equal volume of PBS. Thioglycollate elicited peritoneal macrophages (PM) were labelled with Cell Tracker Green CMFDA (Cell Tracker Green CMFDA Dye; Life Technologies, Carlsbad, CA, USA). Next, labelled PMs were injected retro‐orbitally. Mice were killed after 48 hrs and epididymal fat tissue was harvested, and the stromal vascular fraction (SVF) was isolated. The isolated ATM were stained with Cd11b AlexaFluor700 (eBioscience, San Diego, CA, USA), F4/80 eFluor450 (eBioscience) and Cd45 APC (Biolegend, San Diego, CA, USA) as reported earlier 4.

To examine the infiltrated leukocytes in kidney, single-cell suspensions were prepared as previously described [32 (link)], and stained using the the following fluorochrome-labeled antibodies: anti-CD45 (PE-CY5), CD11b (FITC), Ly6G (APC), and F4/80 (eFluor 450) (eBioscience, CA, USA). Flow cytometry was performed on a FACSCalibur (Becton Dickinson, Heidelberg, Germany), analyzed using FlowJo 10.0 software. Single-cell suspensions of kidney were prepared.
The AnnexinV-FITC/propidium iodide (PI) staining kit was applied to assess cell apoptosis using a flow cytometer (Accuri C6, BD) according to the manufacturer’s instructions. Quadrant statistics on AnnexinV-positive/PI-negative, double-positive cells were performed.

Infected mouse lungs (500 SeV pfu/gram body weight) were mechanically homogenized using the gentleMACS tissue dissociator (Miltenyi). The resulting cell suspension was passed through a 70 μm mesh filter then washed once in ice-cold PBS. Cells (300,000) were incubated with Fc block for 20 min on ice then stained with fluorescent-labeled primary antibodies for 30 min on ice in the dark. CD45-PerCP, NK1.1-APC, Gr-1-FITC and CD3-Alexa Fluor 700 antibodies were purchased from BD Biosciences. The F4/80-efluor 450 was purchased from eBioscience and the CD11b-PE was purchased from Invitrogen. Cells were washed once in ice-cold staining buffer (PBS + 1% BSA and 0.1% sodium azide) then fixed with 1% formaldehyde. Total number of specific leukocyte population per lung was calculated as follows: # of specific leukocyte population = total lung cell number x %gated cells x %single cells x %CD45⁺ cells x %specific leukocyte population. Flow cytometric data were analyzed with FlowJo software (Treestar, Ashland, OR).