Anti mouse cd16 cd32

For live cell/dead cell discrimination, Zombie Aqua dye (BD Biosciences, San Jose, CA) was used according to manufacturer's protocol. Isolated MDLN cells were first blocked with 1% mouse IgG (Sigma-Aldrich) and 1% anti-mouse CD16/CD32 (BD Biosciences), then stained with anti-mouse MHCII-Alexa Fluor 627 (clone M5/114.15.2; BD Biosciences) and anti-mouse CD11c-allophycocyanin-Cy7 (clone N418; BD Biosciences), according to the manufacturer's protocol. Analyses were performed on a FACSCanto flow cytometer (BD Biosciences), and data were analyzed with the FlowJo 7.5.

Single cell suspensions were prepared from aortas as previously described.^{3 (link), 20 (link)} Briefly, the entire aorta with surrounding perivascular fat was minced with fine scissors and digested with 1 mg/mL collagenase A, 1 mg/ml collagenase B, and 100 µg/ml DNase I in phenol-free RPMI 1640 medium with 5% FBS for 30 min at 37°C, with intermittent agitation. Fc receptors were blocked with anti-mouse CD16/CD32 for 20 min at 4°C (BD Biosciences, clone 2.4G2) prior to the staining of surface markers. The antibodies used were: PerCP-Cy5 anti-CD45; PE anti-CD34; APC anti-CD34, APC-Cy7 anti-Sca-1/Ly-6A; PE-Cy7 anti-c-kit/CD117; Amcyan anti-CD31. All aortic cells were incubated with 1.5 µl of each antibody in 100 µl of FACS buffer for 35 minutes. The cells were then washed twice with FACS buffer and immediately analyzed on a FACSCanto flow cytometer with DIVA software (Becton Dickinson). Dead cells were excluded from analysis with a fixable Violet dead cell stain. Intracellular staining was then performed with the Cytofix/Cytoperm™ Plus fixation/permeabilization solution kit (BD Biosciences) using a mouse monoclonal antibody (Abcam) conjugated to Alexa Fluor 488 or 647 (Molecular Probes). For each experiment, we performed flow minus one (FMO) controls for each fluorophore to establish gates. Data analysis was performed using FlowJo software (Tree Star, Inc.).

Flow cytometry was used to detect cell surface receptors and fluorescein isothiocyanate-conjugated LPS (FITC-LPS) associated with intact cells as previously described with minor modifications [40 (link)]. For the detection of cell surface receptors, cells were pretreated with fatty acid as indicated above with or without ultra-pure LPS for stimulation. One million cells were blocked with 1 μg anti-mouse CD16/CD32 (BD Biosciences, San Jose, CA) in 100 μL for 5 min at 4°C and then labeled with 0.25 μg anti-TLR4-APC (R&D Systems), 0.5 μg anti-TLR4/MD2-APC (eBioscience), 0.5 μg anti-CD14-PE (eBioscience), or their isotype controls in 100 μL blocking solution for 30 min at room temperature. To assess the effect of LPS-cell association, fatty acid-treated cells were harvested and suspended in the original culture media containing the treatment fatty acid, and incubated with LPS-FITC (1 μg/mL final concentration) for 1 h at 37°C. Fluorescent labeled cells were washed and resuspended in the staining buffer (R&D Systems), and analyzed on an Accuri Flow Cytometer (BD Biosciences).