Mouse igg2a

MCs and neutrophils (1 × 10⁶ cells/well) were incubated in 24-well plates pretreated with fibronectin (1 mg/well; Life Technologies) for 3 h. Adherent cells were stimulated with 100 µg/ml LPS, 25 nM phorbol-12-myristate-13-acetate (PMA) (MP Biomedicals), or bacteria (bacterium-to-cell ratio of 25 and 50 bacteria per cell for S. aureus and C. burnetii, respectively) at 37°C. The roles of CD36 and TLR4 were studied using MCs pretreated with 5 µg/ml CD36-blocking antibodies (Abs) (mouse IgG2a; Thermo Fisher Scientific) or 10 µg/ml of the TLR4 inhibitor polymyxin B sulfate (Sigma-Aldrich) for 10 min.

The following antibodies were used at 10μg/ml to block integrin function: rat IgG2a anti-human CD29/β1-integrin (Mab13; BD Pharmingen); mouse IgG2a anti-human CD18/β2-integrin (IB4; Calbiochem); mouse IgG1 anti-human CD61/β3-integrin (SZ21; Beckman Coulter); matched control antibodies, rat IgG2a, mouse IgG2a (both eBioscience), mouse IgG1 (DAKO). In some experiments, RGDS peptide (Arg-Gly-Asp-Ser, 0.5 mM; Sigma) or CT7010 (20 μM) was used instead of antibody. CT7010 is a low molecular weight, non-peptide, inhibitor of β₂-integrin function (gift of Dr. Tony Shock, Celltech R&D) The above antibodies and inhibitors have previously been shown to block functions [11 (link),40 (link),41 (link)].
Neutrophils were treated with antibodies for 10 min prior to addition to the gels. For endothelial monolayers, after 10 min the non-adherent cells were removed by washing with M199/BSA (0.15%) and 1 ml of M199 BSA (0.15%) containing the agent was re-added to the gel. In separate experiments, agents were added after 15min when initial migration had occurred. We showed that these added antibodies did reach and bind to integrins within 1h by retrieving cells, labelling them with secondary anti-integrin and flow cytometry (see below). Neutrophils were also treated with antibodies 10min before analysis in the phagocytosis assays.

Human MBL protein was purchased from ACRO Biosystems (MBL-H5220), and the purity > 95% as determined by SDS-PAGE. PGN was purchased from Sigma (72789) with the purity > 95%, which is isolated from Saccharomyces cerevisiae. Anti-MBL polyclonal antibodies (R&D Systems, MN, USA), anti-TLR2 antibody (clone TL2.1, eBioscience, San Diego, CA), and mouse IgG2a were purchased from eBioscience. The specific antibody for IκB-α, p65, phospho-IκB-α, p38 MAP kinase, c-Jun N-terminal kinase (JNK), phosphorylated ERK1/2, phospho-p38 MAP kinase, phosphorylated JNK and secondary rabbit anti-mouse IgG-HRP antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Actin, Histone H1, goat anti-rabbit IgG-HRP, and donkey anti-goat IgG-HRP antibody were purchased from Santa Cruz Biotechnology.

Peripheral blood samples were collected with Li-Heparin Monovettes (Sarstedt) and processed within 3 h. Red blood cells were eliminated by osmotic lysis with RBC lysis buffer as described before [2 ]. Leucocytes were then washed, re-suspended in FACS buffer (DPBS + 10% FCS). Unspecific binding of antibodies to cell surface receptors was blocked with Fc Block (1:20, BioLegend) or human TruStain Fc Block (BioLegend) for 20 min at 4 °C. Leucocytes were stained with antibodies specific for CD3 (1:50, BioLegend; UCHT1-PB), CD4 (1:100, BioLegend RPA-T4-PE), CD8 (1:100, BioLegend, SK1-APC), and CD69 (1:50, BioLegend; FN50-FITC) for 25 min at 4 °C in the dark. After washing, the stained cells were fixed for 20 min in 2% PFA, filtered through a 70 µM cell-strainer and characterized by flow cytometry on a LSRII flow cytometer (BD) using a combination of unstained cells, internal negative population controls and single-color staining for color compensation. Therefore, isotype-matched, host-matched monoclonal antibodies were used as negative control: mouse IgG2a, κ (BioLegend; MOPC-173-PE-Cy7), mouse IgG1, κ (BioLegend; MPOK-21-PB), mouse IgG1, κ (BioLegend; MOPC-21-PE), mouse IgG2a, κ (eBioscience; P3.6.2.81-APC). Flow cytometry data was processed with FACS Diva™ software (BD).

Retinas were collected and digested in the same way as the steps previously mentioned. Immediately after the termination of digestion, the centrifuge tube containing the digested retina was cooled in ice and then centrifuged at 500×g at 4°C for 5 min. After the supernatant was discarded, the pellet was resuspended in 500 μL of precooled HBSS. Subsequently, the resuspended cell suspension was centrifuged at 800×g at 4°C for 5 min; and repeated step 1 time. After the supernatant was discarded again, the pellet was resuspended in 100 μL of flow buffer (eBioscience, USA). The classification of gating was based on Isotype Control which was equivalent to the negative control of the experiment. Thus, we chose Mouse IgG2A for gating. The flow antibody anti-rat CD11b/c (0.125 μg/tube, Mouse IgG2A, eBioscience) was added to the sample tube; the flow antibody Mouse IgG2A (0.125 μg/tube, eBioscience, USA) was added to the control tube. After mixing gently, the tubes were placed in the dark at 4°C for 30 min, and then centrifuged at 4°C and 300×g for 5 min. After the supernatant was discarded, the stained cell pellet was resuspended in 100 μL of flow buffer and centrifuged at 4°C and 300×g for 5 min, and this step was repeated once more. The stained cell pellet was resuspended in flow buffer, and then placed on ice before analyzed with flow cytometry.

The following FITC- or PE-conjugated mAbs were purchased from BD Pharmingen (San Diego, CA), eBioscience (San Diego, CA), R&D Systems (Minneapolis, MN), Abcam (Bristol, UK) or Miltenyi Biotec (Bergisch Gladbach, Germany): anti-HLA class II (clone TU39, Mouse IgG2a), anti-HLA class I (clone G46-2.6, mouse IgG1), anti-CD80 (clone L307.4, mouse IgG1), anti-CD83 (clone HB15e, mouse IgG1), anti-CD86 (clone FUN-1, mouse IgG1), anti-CD40 (clone 5C3, mouse IgG1), and anti-CD8 (clone T8, mouse IgG1). Mouse IgG2a (clone G155-178), mouse IgG2b (clone 27–35), mouse IgG1 (clone MOPC-21), affinity purified mouse IgM (eBioscience), and rat IgG2a (clone eBR2a) were used as isotype-matched controls. The cell samples were treated with an Fc-receptor-blocking reagent (Miltenyi Biotec) for 10 min, stained with the fluorochrome-conjugated mAb for 30 min, and washed 3 times with PBS containing 2% FCS. The stained cell samples were analyzed on a FACScan (BD Biosciences, Bedford, MA) flow cytometer.