Fcr blocking agent

Mice were sacrificed at 3 or 8 dpi, and the lung lobes were fixed, dehydrated and frozen. Freshly cut lung sections (5 μm thickness) placed on poly-L-lysine-coated slides were pretreated with 1:10 FcR blocking agent (Miltenyi Biotech, Gladbach, Germany) for 10 min and reacted with various antibodies as follows: goat anti-prosurfactant protein (proSP)-C antibody (Santa Cruz Biotech., Santa Cruz, CA), rabbit anti-phospho-Stat3 (Cell Signaling Technology), rabbit anti-phospho-Akt antibody (Cell Signaling Technology), rabbit anti-Iba 1 antibody (WAKO, Osaka, Japan), rat anti-mouse IL-6 antibody (Biolegend, San Diego, CA), rabbit anti-S100A4 antibody (Abcam, Cambridge, UK) or mouse anti-smooth muscle α-actin/SMA (Sigma-Aldrich, St. Louis, MO). After staining with each appropriate fluorescein-conjugated second antibody, the sections were observed under a fluorescence microscope (Axio Imager A2, Zeiss, Oberkochen, Germany). Nuclei were stained with 4’6’-diamino-2-phenylindole (DAPI).

The physicians used real-time X-ray technology to visualize the patient’s vascular system and locate IA inside the blood vessel. A solitaire stent retriever (Covidien, Irvine, CA, United States) was used for biopsy specimens’ retrieval from an occluded IA. Cerebral aneurysm and malformed arteries were isolated from cerebral arties using a cutting plane and separating the surfaces on either side of the plane by using a minimally invasive technique. Endothelial cells were derived from human cerebral aneurysm and malformed arteries, and separated with immunomagnetic cell sorting. Briefly, Anti-CD 146-coated Dynabeads (Invitrogen, CA, United States) were prepared according to manufacturer’s instruction and stored at 4°C. Fifty-mg aneurysms were ground with glass pestle and mortar with one-millilter PBS buffer, 0.1% bovine serum albumin, 0.1% sodium azide, and 0.1% a standard broad-spectrum inhibitor cocktail at 4°C. Ten-microliter FcR-blocking agent (Miltenyi, Bergisch Gladbach, Germany) and 25-microliter antibody-coated Dynabeads were added and mixed thoroughly. The samples were mixed in a mixer for 1 h at 4°C and washed four times with PBS inside the Big Easy Magnet (EasySep, United States) at 4°C. Between each washing procedure, the endothelial cells were flushed out with MACS buffer (PBS with 0.5% BSA and 2 mM EDTA, pH 7.0) 10 times in a 100-μL pipette.

The phenotype of hESC-MSCs was characterized using flow cytometry and the following monoclonal antibodies: anti-CD31-APC (Miltenyi Biotec), anti-CD44-FITC (BD Pharmingen), anti-CD45-FITC (BD Pharmingen), anti-CD73-APC (Miltenyi Biotec), anti-CD90-FITC (BD Pharmingen), anti-CD105-PE (eBioscience), HLA-ABC-APC (BD Pharmingen), and HLA-DR-FITC (BD Pharmingen). Briefly, the cells were dissociated and suspended in FACS buffer (1x PBS/0.5% BSA) and nonspecific binding blocked with FcR blocking agent (Miltenyi Biotec) for 10 minutes at 4°C. For labeling cell surface antigens, the cells were incubated with the abovementioned fluorescent conjugated antibodies for 10 minutes at 4°C. After antibody labeling, data was acquired using Dako Cytomation CyAn ADP and analyzed using FlowJo v7.6.5 (Tree Star).

Specific markers of PBMCs and monocytes were labeled and analyzed by flow cytometry as we previously described.²⁴ Briefly, after sterilization with alcohol, spleens were isolated from treated Balb/c mice. Subsequently, the spleen samples were at 70 μm by nylon cells mesh (BD Falcon, MA, USA). A red blood cell separation buffer (R&D Systems) was used to remove red blood cells. An FcR blocking agent (Miltenyi Biotec) was adapted to exclude nonspecific antibody combination in PBMCs and monocytes cell culture. Incubating together with the suitable antibody for half an hour at 4°, the cells were washed with FACS buffer and passed through a BD LSR II (BD Biosciences, San Diego, CA). The cells were incubated with anti‐CD4 FITC PerCP for 1 hour, subsequently being fixed and permeabilized by a fixation/permeabilization kit (BD Biosciences). Then, the cells were stained with anti‐Foxp3 and analyzed by flow cytometry. A FlowJo software (Tree Star) was used to collect and analyze the data.