Anti cd11b pe cy7

Isolated cells in the lung were stained with indicated antibodies for further analysis. For immunostaining, the cells were washed two times with PBS containing 2% FBS, adjusted to approximately 1 × 10⁵ to 1 × 10⁶ cells in 100 μL of the same buffer, and labeled with PE-anti-F4/80 (BD Bioscience, 565410), PE-Cy7-anti-CD11b (BD Bioscience, 561098), and FITC-anti-TNF-α (BD Bioscience, 554418). Incubations with antibodies were performed for 30 min at 4 °C in the dark, and then cells were centrifuged for 4 min at 900 rpm. After removal of the supernatant, the cell pellet was washed once with PBS containing 2% FBS and then resuspended in fixation and permeabilization buffer (BD Bioscience, Franklin Lakes, NJ, USA) for 30 min at 4 °C. Cells were washed two times with PBS containing 2% FBS and then acquired by FACSVerse (BD Bioscience) and analyzed using software Flow Jo (Tree Star, Inc., Ashland, OR, USA).

Isolated single cells were resuspended in flow cytometry buffer (2% BSA, 10μM HEPES in HBSS without calcium/magnesium) at approximately 10⁷ cells/mL and processed at 4 °C. Cells were blocked with mouse IgG isotype control (ThermoFisher) and rat IgG isotype control (R&D Systems) as appropriate. Cells were then incubated with the appropriate conjugated primary antibodies (FITC-anti-CD45, BD Pharmigen 555482; PE-Cy7-anti-CD11b, BD Pharmigen 557743; PE-anti-CD3, Biolegend 300308; APC-Cy7-anti-CD31, Biolegend 303120) for 1 h. Next, cells were stained using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) according to the manufacturer’s instructions. Immediately before analysis and sorting, DAPI was added to the flow cytometry buffer. Cells were sorted and analyzed on a BD FACSAria II or FACSAria Fusion. Cells were identified and gated for size, singularity, cell viability, and all surface-marker expression was gated with appropriate full-minus-one controls. Cells were sorted into cold catching medium and a fraction was removed for cell purity analysis. Isolated cells were lysed in TRIzol (Invitrogen), and stored at − 80 °C until further processing.

Mice were immunized i.p. with 25 μg/animal of CpG ODN 1826 (InvivoGen) or with 5 μg/animal of Salmonella flagellin (FliC). 6 h after immunization, mice were euthanized and splenocytes were labeled. Fc receptors were blocked with Fc Block (BD Biosciences) and subsequently stained first with anti-CD19-Biotin (clone 1D3), anti-CD3-Biotin (clone 145.2C11), and anti-CD49b-Biotin (clone DX5) for 40 min on ice. After two washes with PBS-2% FBS, cells were then incubated anti-MHCII (I-A/I-E)-Alexa Fluor 700 (clone M5/114.15.2), anti-CD11c-BV421 (clone N418), anti-CD11b-PE.Cy7 (clone M1/70), anti-CD8α-BV786 (clone 52–67), anti-CD80-FITC (clone 16-10A1), anti-CD86-APC (clone GL1), anti-CD40-PE (clone 1C10), Streptavidin APC.Cy7 (all antibodies and the streptavidin were purchased from BD Biosciences) and Live and Dead Aqua (Life Technologies). Flow cytometry was performed using LSRFortessa (BD Biosciences) and results were analyzed in FlowJo software (version 9.3, Tree Star, San Carlo, CA, USA).

In order to investigate targeting ability of the antibody in tissue condition, we first performed near-infrared fluorescent imaging on excised aortas. Briefly, anti-CD11b-PE-Cy7 (BD Bioscience) was injected to 5 ApoE^−/− and 5 C57 mice via tail vein (1 mg/kg), while IgG-PE-Cy7 (BD Bioscience) with same dose was injected to another 5 ApoE^−/− mice. Twenty four hours later, mice were sacrificed. NaCl (0.9%, 5 mL) and paraformaldehyde (4%, 5 mL) were sequentially injected into vascular system to eject blood and fix vessels. Then, aorta was carefully dissected, placed on a microscope slide, covered with gauze (rinsed with 0.01 M PBS), and imaged within 1 h in the Carestream FX PRO (Kodak, USA). The Cy7 label was excited using a wavelength filter of 615–665 nm, while the emission was recorded using a 695–770 nm filter. Fluorescent images were acquired with identical exposure time for all aortas. Area percentage of plaque was measured and total photon count was recorded around plaque areas. Mean signal intensity was calculated by dividing the total photon count to the total area of plaques (SI-mean). Likewise, noise intensity (NI-mean) was measured by drawing a ROI in normal area of the aorta. Finally, a signal-to-noise ratio (SNR) was calculated as: SI-mean/NI-mean.