Hla dr

The Paraffin-embedded specimens were sectioned at 3 μm thickness. After dewaxing, antigen retrieval was made in the microwave of citrate buffer. The slices were then blocked with 3% BSA and stained overnight at 4 °C with the primary antibodies against human Wnt5a (1:100, Abcam), CD68 (1:100, Abcam), HLA-DR (1:100, Abcam), CD163 (1: 100, Abcam), IL-10 (1:100, Abcam). Subsequently, specimen sections were incubated with fluorochrome-conjugated secondary antibodies. Nucleus staining was performed using Diaminophenylindole (DAPI).
Before immunofluorescence staining, all samples were stained with hematoxylin and eosin (H&E) to ensure that all specimens contained representative CRC tissue. For each section, at least 6 fields of view (at 200× magnification) were used to calculate positive cells stained with indicated antibody. A quantitative analysis was performed to determine the numbers of infiltrating CD68⁺ and Wnt5a⁺CD68⁺ cells. All of the above-mentioned cells were counted manually. Only cells with macrophage-like morphology were included. Cell counting was carried out in a blinded manner by two research fellows familiar with histomorphology and immunofluorescence staining. If there was a disagreement, the analyses would be discussed with the authors.

Bone marrow mesenchymal stem cells (BMSCs, Institute of Hematology, Peking University People's Hospital, Beijing, China) and human placental mesenchymal stem cells (HPMSCs, Boya Stem Cell Bank, Beijing, China) were placed in a complete medium of DMEM/F-12 (GIBCO, USA) containing 10% of fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (GIBCO, USA), before being transferred to a cell culture incubator. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed once every two days (2 d). The cell growth status was observed under a microscope. When the confluence density of the cells reached 80–90%, they were passaged using routine subculture techniques, and some cells were cryopreserved at the same time. The cells were passaged continuously, and subsequent experiments were performed using P6–P9 generations of the cells. The content of MSC characterization included induction of adipogenic differentiation, induction of osteogenic differentiation, and the presence of surface markers of MSCs including CD105, CD73, CD34, CD11b, CD19, CD45, and HLA-DR (Abcam, USA) (Figure 1(a)).

The MSC immunophenotype was determined by flow cytomety using the Navios Cytometer (Beckmann Coulter, IN, USA) and FlowJo analysis software (version 7.4.1. TreeStar Inc., CA, USA). The cell surface antigens CD44, CD49e, CD13, CD90, CD73, CD29, CD86, CD105, HLA-ABC, CD14, CD19, CD34, CD45, CD80, and HLA-DR were analyzed and at least 5000 cells were included in the analysis. All antibodies were purchased from BD Biosciences (CA, USA) except CD80 (Beckmann Coulter) and HLA-DR (Abcam, UK). The proportion of non-viable cells were determined during analysis by staining the cells with propidium iodide (50 μg/ml in PBS, BD Biosciences).

hUCMSCs immunophenotypes were evaluated. Harvested cells were labeled using mouse anti-human monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated CD105 (20 μl/10⁶ cells), CD90 (5 μl/10⁶ cells), CD73 (5 μl/10⁶ cells), CD14 (20 μl/10⁶ cells), CD34 (10 μl/10⁶ cells), CD45 (10 μl/10⁶ cells), and HLA-DR (1 μl/10⁶ cells) (Abcam Inc., MA, USA). FITC-labeled mouse anti-human immunoglobulin G1 (IgG1) was used as the homotype control. MSC phenotypes were identified using a flow cytometer (BD Biosciences, NJ, USA).

The proteins from the cells were resolved on SDS-PAGE gels, transferred onto nitrocellulose membranes, and immunoblotted with antibodies against HLA-DR, ICAM-1, CD80, IL-1α, IL-6, NFκB, CCL2, and β-actin (Abcam, Cambridge, MA, USA). Secondary antibodies were then applied, and the signals were detected using a ChemiDoc-It 600 Imaging System (UVP, Upland, USA).