Anti cd11b apc

The principle of the β-lactamase CCF-4 FRET assay is summarized in S1 Fig.. To
measure the Mtb phagosomal rupture, cells were stained during 1h at
RT, with 8 μM CCF-4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM
CaCl₂, 0.8 mM MgCl₂, 5 mM glucose and 25 mM Hepes, pH 7.3)
complemented with 2.5 μM probenecid. Cells were then stained with
anti-CD11c-PE-Cy7, anti-CD11b-PerCp-Cy5.5 (eBiosciences) or anti-CD11b-APC (BD) mAbs
andfixed with 4% PFA overnight at 4°C. Cell mortality in the same cultures of
infected cells was determined by use of Pacific Blue Dead/Live reagent (Invitrogen),
which reacts with free amines both inside and outside of the plasma membrane,
yielding log₁₀ 1 more intense fluorescent staining of dead cells.
Anti-CD45.1-PE-Cy7 and anti-CD45.2-PerCpCy5.5 were from eBiosciences. To avoid
fluorochromes with emission signals overlapping with those of CCF-4
(λ_em 500–550 nm and λ_em 410–470
nm), APC (λ_em 660 nm)-, PerCp-Cy5.5 (λ_em 696 nm)-
or PE-Cy7 (λ_em 778 nm)-conjugated mAbs were chosen for concomitant
cell surface staining. Cells were analyzed in a CyAn cytometer using Summit software
(Beckman Coulter, France). At least 100,000 events per sample were acquired for
in vitro assays. For in vivo detection of CCF-4
signal in CD45 congenic mouse model, 1,000,000 events per sample have been acquired.
Data were analyzed with FlowJo software (Treestar, OR).

The mice were euthanized at 4 weeks after MI. Their blood, spleens, bone marrow and infarcted hearts were collected and made into single-cell suspensions for flow cytometry analysis, as previously described10 (link). The cells were stained with a mixture of antibodies (anti-CD11b-APC and anti-Gr-1-PerCP-Cy5.5 BD Biosciences). The data were acquired using an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo7 software (Tree Star, Inc.).

Mice were sacrificed at 1 day and 7 days after MI. The blood, spleen, bone marrow and infarcted myocardium were collected and made into single-cell suspensions for flow cytometry, as previously described26 (link)52 (link). The cells were stained with a mixture of antibodies (anti-CD11b-APC, anti-Gr-1-PerCP-Cy5.5, anti-Ly6C-PE; BD Biosciences). Data were acquired using an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo7 software (Tree Star, Inc.).

FACS analysis was performed according to the previous reports (22 (link)). After perfusion of the kidneys with cold 1 × PBS, kidneys were removed, cut into fragments, and digested with 1 mg/ml collagenase and 0.1 mg/ml DNAase for 1 h at 37°C with intermittent agitation. Kidney fragments were passed through a 70-μm mesh (FALCON, 352350), producing single-cell suspensions. Approximately 1 × 10⁶ cells were stained for 30 min at room temperature with antibodies including anti-Mouse CD45-PE (8205729; Invitrogen), anti-CD11b-APC (8278517; BD Pharmingen), and anti-Mouse CD206-PE (4336334; BD Pharmingen), and resuspended in 1 × PBS. The suspensions were washed thrice with 1 × PBS, resuspended in 1 × PBS, and analyzed on High configuration Analysis Flow Cytometry (BECKMAN, CytoFLEX LX).

The DRAG targeting construct was linearized using PvuI and electroporated into IB10 E14 129/ola ES cells. Stable transfectants were selected with puromycin, and resistant clones were picked and expanded. Correct integration was determined by Southern blotting, using a probe directed against the 5′ Rosa26 homology arm. Two independent ES cell clones were injected into C57Bl/6 blastocysts to generate 28 transgene-positive chimeric mice and establish two independent DRAG transgenic lines (DRAG1 and DRAG2). GFP expression in peripheral blood B and T cells was screened using anti-CD19-PE (BD, clone 1D3, dilution 1/100), anti-CD3e-PerCP-Cy5.5 (eBioscience, clone 145-2C11, 1/100), and anti-CD11b-APC (BD, clone M1/70, 1/100), and the DRAG1 line was selected. DRAG1 mice were crossed with B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (CAGGCre-ER^TM) to obtain heterozygous mice for experimental use.

Mice were injected intraperitoneally with 100 μL PBS, or 5 μg kg⁻¹ IL‐33 (PeproTech) or 2.5 mg kg⁻¹ LPS (Escherichia coli strain O26:B6; Sigma; L2654) in 100 μL PBS. After 3 h, the mice were sacrificed via an increasing concentration of CO₂, and the peritoneal cavity was washed using 4 mL PBS containing 1% BSA and 5 mm EDTA. Cells were pelleted by centrifugation and incubated on ice with Fc Block (1 in 50 dilution, BD Biosciences) for 10 min. Cells were then stained with anti‐Gr1‐PerCp‐Cy5.5 (1 in 800, BD Biosciences, #552093) and anti‐CD11b‐APC (1 in 1600, BD Biosciences #553312) for 30 min. Cells were then washed twice and analyzed by flow cytometry. For analysis, live cells were gated based on a forward and side scatter and neutrophils defined as CD11b Gr1 double‐positive cells. Gating and representative FACS plots are shown in Supplementary figure 12.

The stromal visceral fat fraction (SVF) and spleen were used for the analysis of major histocompatibility complex class II (MHCII) cells. For this purpose, the obtained fraction was stained with anti-MHCII-PercpCy5.5 (562363 BD), anti-CD11C-PE (117308 BioLegend), anti-CD80-FITC (553768 BD), and anti-CD11b-APC (553312 BD); washed; and resuspended in PBS-5% FBS. The samples were analyzed using a FACSCanto II analyzer (BD Bioscience). Data were analyzed using FlowJo software for flow cytometry analysis.