Anti cd16 cd32 mab fc block

GFP-Pc iRBC-infected B6 mice were sacrificed and PBS-perfused. Spleens were removed and frozen in Tissue-Tek OCT (Sakura Fineteck, Japan). Sections 8 µm thick were cut with a CM3050S Cryostat (Leica, USA) and fixed with 1% paraformaldehyde (Alfa Aesar, USA) for 30 min at RT. Sections were incubated with anti-CD16/CD32 mAb (Fc block; BD Biosciences) for 30 min followed by incubation in a humidified dark chamber with fluorescent mAbs against CD11c, CD19, CD3, CD4 (BD Biosciences) and MOMA-1 (Abcam) for 2 h at RT. Sections were then stained for 5 min with 0.5 μg/ml DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich), washed with PBS and mounted with Fluoromount-G (Southern Biotechnologies, USA). Images were acquired with a DMRA2 fluorescence microscope (Leica) and MetaMorph software (Molecular Devices Inc., USA). Image analysis was performed with Photoshop CS4 (Adobe Inc., USA). Percentages of CD11c-GFP/CD11c-CD4 pixel colocalization and of GFP pixel distribution in the spleen were calculated using FIJI for Windows 64-bit (Colocalization threshold and Mixture Modeling Thresholding plugins, respectively; General Public License, NIH, USA).

Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI medium, fetal calf serum (FCS), glutamine, gentamicin, sodium pyruvate, MEM nonessential amino acids, and HEPES buffer were obtained from GIBCO/Invitrogen. The following reagents were used: Nutridoma SP (Roche); propidium iodide, DMSO, PE-conjugated Annexin V, hamster IgG1/κ, PE- and FITC-conjugated hamster anti-mouse Fas and FasL monoclonal antibodies (mAbs), APC/Cy7-conjugated anti-Ly6G, purified hamster anti-FasL MFL3 mAb, hamster IgG1 isotype control, anti-CD16/CD32 mAb (FcBlock), all from BD Biosciences; PerCP/Cy5.5-conjugated anti-Ly6C mAb HK1.4 (eBioscience); rabbit SAPK/JNK mAb 56G8; phospho-SAPK/JNK (Thr183/Tyr185) mAb 81E11; anti-c-Jun mAb 60A8, phospho-c-Jun-Ser63 mAb 54B3, Goat anti-rabbit IgG, HRP-conjugated (Cell Signaling); JNK inhibitor SP600125 (Enzo Life Sciences), ERK (MEK) inhibitor PD98059, p38 inhibitor SB203580 (EMD Millipore).

Freshly isolated mouse lung endothelial cells (MLECs) were obtained from lungs as described (Oblander et al, 2005). Red blood cells were removed by incubating the lung cell suspension for 5 min in ACK buffer (150 mM NH₄Cl, 0.1 mM Na₂EDTA, 10 mM KHCO₃, pH = 7.4). The cells were then resuspended in sorting buffer (PBS + 1% FBS + 10 mM HEPES) and incubated for 10 min at 4°C with an Fc‐block anti‐CD16/CD32 mAb (Pharmingen) to block non‐antigen‐specific binding of immunoglobulins to the Fcγ receptors. This was followed by staining with Alexa Fluor 488‐conjugated anti‐mouse CD31 (Mec13.3) and V405‐conjugated anti‐mouse CD45.1 (all from BD Bioscience Pharmingen). The endothelial cell population defined as CD31⁺/CD45⁻ was sorted in a Synergy 4L Cell Sorter (Sony Biotechnology Inc.), and MT1‐MMP deletion efficiency was analyzed by qPCR.