Periodontal tissues were obtained from the extracted third molar teeth at the department of Oral and Maxillofacial surgery in the First Affiliated Hospital of Hainan Medical University. Totally 24 teeth used in the experiment were extracted from young men between the ages of 20 and 30. The teeth were free from infection, and patients were free from systemic diseases. The study was carried out under ethical guidelines and with the consent of each patient involved in the study. The tissue cleaner was PBS containing 10% FBS. The primary cell culture medium was DMEM containing 10% FBS. Basal medium of STEMPRO osteocyte contained 10% osteogenesis supplement. The cell digestion solution was 0.25% EDTA trypsin. All of the above cell culture reagents were purchased from GIBCO. Fluorescence direct labeling antibodies including STRO-1, CD146, CD34 and CD45 were purchased from the Abcam company. Primary antibody of RAGE and HRP labeled second antibody were purchased from SANTA CRUZ and CST, respectively. AGE-BSA was purchased from CALBIOCAM, and carboxy-H2DCFDA ROS Kit was purchased from Invitrogen.
Cd146
Manufactured by Abcam
Sourced in United States, United Kingdom
CD146, also known as MCAM, is a cell surface glycoprotein that is expressed on endothelial cells, pericytes, and some types of cancer cells. It plays a role in cell-cell adhesion and migration.
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Lab products found in correlation
Cd105, by Abcam (3 mentions)
Stro 1, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Stro 1, by Abcam (1 mentions)
Canto 2, by BD (1 mentions)
P pak1 t423, by Santa Cruz Biotechnology (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
F4 80, by Abcam (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Ab181597, by Abcam (1 mentions)
Ab75769, by Abcam (1 mentions)
Coverslip, by NEST Biotechnology (1 mentions)
Fitc labeled goat anti rabbit igg, by Beyotime (1 mentions)
Cy3 labeled goat anti rabbit igg, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Eclipse ni, by Nikon (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Aggrecan, by Abcam (1 mentions)
Cd166, by Abcam (1 mentions)
Dmem f12 cell culture medium, by Thermo Fisher Scientific (1 mentions)
15 protocols using cd146
1
Isolation and Characterization of Periodontal Stem Cells
2
Mesenchymal Stem Cell Phenotyping by FACS
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Cells grown to an optimal confluence of 70% were used for FACS analysis. Single cell suspension was prepared by dissociating the cells using 0.05% Trypsin and triturating in PFN (PBS + 5%FBS + 0.1%Sodium Azide). The cells were fixed with 4% paraformaldehyde in PFN over ice for 1 hour. The fixed cells were incubated with two primary antibodies per experiment (CD29[Abcam, 1:100]/CD146[Abcam, 1:50 and CD44[Abcam, 1:50]/Stro1[R&D Systems, 1:50]) at 4 °C overnight. Subsequently, the cells were incubated with fluorescent secondary antibodies over ice for 1 hour and analysed by flow-cytometer (Canto II, BD Biosciences). FlowJo software was used for gating and further analysis.
3
Immunohistochemical Analysis of RCC Tumor Markers
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Tumors sections from subcutaneous tumor xenografted nude mice and patients with RCC were immunohistochemically analyzed as described previously.60 (link) Primary antibodies against IL-6, CD73, CD146 (Abcam, Cambridge, MA, USA), PAK1 and p-PAK1 (T423) (Santa Cruz Biotechnology) were used in the procedure.
4
Comprehensive Histological Assessment of Tissues
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Wound, liver, and spleen tissues were embedded in paraffin. Multiple 5 μm sections were stained with hematoxylin and eosin (H&E) (Sigma‐Aldrich) for general histological analysis; F4/80 (1:100, Abcam) for inflammation, proliferating cell nuclear antigen (PCNA) (1:1000, Abcam) for proliferation; CD146 (1:100, Abcam) for anagenesis and Picro‐Sirius red staining with polarized imaging for collagen deposition.
5
Evaluation of Mesenchymal Stem Cell Markers
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Expressions of specific antigen on the rMSC surface were identified by flow cytometry analysis (FACS). Passage 3 rGMSCs were incubated at 4°C in the dark room for 30 min with FITC-conjugated monoclonal antibodies against rat STRO-1 (eBioscience, USA), CD34 (Abcam, UK), CD105 (Abcam), CD146 (Abcam), CD73 (Becton-Dickinson, USA), and CD90 (BioLegend, USA), with FITC-conjugated mouse anti-rat CD45 (Becton-Dickinson) and with FITC-conjugated rabbit CD14 (Bioss, USA). After thorough washing with PBS, cells were subjected to flow cytometry analysis to evaluate the proportion of expressions with the use of a FACScan flow cytometer (Becton-Dickinson).
6
Immunostaining of Mesenchymal Stem Cells
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P4 MenSCs were cultured on coverslips (14 mm, NEST) at 5 × 104 cells/ml in 12-well plates. After overnight starvation, the medium was exchanged to 10% PRP or 10% FBS-DMEM/F12 incubated for 24 h. Then the coverslips were washed twice by PBS and fixed in 4% PFA, followed by protein block (SP-9001, ZSGB-BIO) for 30 min at 37 °C. Purified SUSD2 antibody (1:100, #327401, BioLegend), CD146 antibody (1:100, ab75769, Abcam, Cambridge, MA, USA), Vimentin antibody (1:100, D21H3, CST), cytokeratin 18 (CK18) antibody (1:100, ab181597, Abcam) diluted in PBS were immunostained overnight at 4 °C. Next day, all the coverslips were incubated with secondary antibodies (1:500, Cy3-labled goat anti-mouse IgG, FITC-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-rabbit IgG, Beyotime, Beijing, China) at room temperature for 2 h. DAPI (1:20, Beyotime) was used to visualize nuclei. Images were observed and captured using Nikon ECLIPSE Ni (Nikon), the fluorescence was analyzed by DOI/Area using Image Pro-Plus 6 (Media Cybernetics).
7
Characterization of hPDLSCs by Flow Cytometry
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After the removal of the culture medium, the third generation of passage cultured hPDLSCs were collected and washed with PBS. Next, the cells were soaked in PBS buffer containing 0.25% trypsin for 5 min to make a single cell suspension. Then, the cells were washed with PBS solution containing 1% bovine serum albumin, and the cell concentration was adjusted to 1 × 106 cells/mL, followed by incubation with monoclonal antibodies CD146 (Abcam, UK), CD90 (Abcam, UK), STRO-1 (RampD systems, USA) and CD45 (Abcam, UK) for 30 min at 4 °C in the dark. Again, cells were washed with PBS, centrifuged at 1000 g for 5 min and resuspended in PBS. Lastly, the background marker was determined using isotype control monoclonal antibody, and the positive rate (%) of cell surface antigen was analyzed by flow cytometry combined with special supporting software.
8
Mesenchymal Stem Cell Nanoparticle Encapsulation
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BMSCs cells were purchased from Beijing Beina Chuanglian Biotechnology Institute (China). DMEM medium, DMEM/F12 cell culture medium, and FBS were purchased from Gibco (United States of America). Fe3O4 solution, O-carboxymethyl chitosan octadecyl quaternary ammonium salt (OQCMC), dioleoyl Phosphatidylcholine (DOPC), and dimethyloctadecyl epoxypropylammonium chloride (GHDC) were purchased from Xi'an Kaixin Biotechnology Co (China). PEG-modified distearoyl phosphatidylethanolamine (DSPE-PEG) was purchased from Chongqing Yuyin Pharmaceutical Technology Co (China). Methylene chloride, cholesterol, N-hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Shanghai Kether Chemical Technology Co (China). CD105, CD73, CD45, HLA-DR, recombinant sex-determining region y box protein 9 (Sox9), CD90, Aggrecan, Collagen II antibodies, CD34, CD166, and CD146 were purchased from Abcam (United Kingdom). BCA Protein Concentration Assay Kit was purchased from Beyotime (China). Hematoxylin Stain was purchased from Wuhan Xavier Biotechnology Co (China). Eosin stain was purchased from Zhuhai Beyotime Biotechnology Co (China).
9
Characterization of SCAP Cell Surface Markers
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SCAPs were purchased from Shanghai Anwei Biotechnology Co., Ltd. (Shanghai, China). The cultures of SCAPs were described in our previous work.55 (link) SCAPs were cultured as previously described to characterize the cells, they were aliquoted into sterile tubes, and then, anti-human antibodies including CD90 (cat no. ab225; Abcam), CD105 (cat no. ab11414; Abcam), CD146 (cat no. ab75769; Abcam), CD34 (cat no. ab81289; Abcam), and CD45 (cat no. ab10558; Abcam) were added at a 1:500 dilution to each tube. The tubes were incubated at 4 °C for 1 h in the dark. Following three washes with 2% fetal bovine serum (FBS) in PBS, the cells were further incubated with secondary antibodies in the dark for 1 h. Subsequently, flow cytometry was employed to detect cell surface markers.
10
Mesenchymal Cell Immunophenotyping Protocol
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Example 22
SKP-derived mesenchymal cells were detached by trypsinization, and aliquots of 1.0×106 cells were prepared in 5 mL round-bottom test tubes. After rinsing with PBS containing 0.2% FBS, the cells were centrifuged and blocked with PBS containing 1% BSA and 0.2% FBS at 4° C. for 30 minutes. The cells were incubated with primary antibodies to CD29 (1:50; Chemicon), CD44 (1:25; BD Pharmingen), CD73 (1:40; BD Pharmingen), CD133 (1:50; Abcam), CD146 (1:1000; Abcam), and Stro-1 (1:17; Santa Cruz Biotechnology) on ice for 1 hour. After washing with PBS containing 0.2% FBS, the cells were incubated with fluorescein isothiocyanate-labeled secondary antibodies on ice for 1 hour. Finally, the cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson).
The details confirmed by the Examples above are summarized in the following Experimental Examples.
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