Flow cytometry was performed by staining with Zombie Yellow viability dye, blocking with anti-CD16/32, and staining for 30 min at 4°C with the following anti-human antibodies: CD45 (BD:HI30), CD3 (BD:UCHT1), CD11b (Biolegend:M1-70), CD206 (Biolegend:15-2), CD163 (Biolegend:GHI/61), CD271 (Biolegend:ME20.4), PD-L1 (Biolegend:29E.2A3); or anti-mouse antibodies: CD45 (Biolegend:30-F-11), CD11b (Biolegend:M1-70), F4/80 (Biolegend:F4/80), Ly6G (Biolegend:1A8), Ly6C (Biolegend:HK1.4), IA/IE (BD:M5/114), CD206 (Biolegend:C068C2), PD-L1 (Biolegend:10F.9G2), PD-1 (BD:J43), IFN-γ (Biolegend:XMG1.2), CD3 (Tonbo:145-2C11), CD8 (Biolegend:YTS156.7.7), CD4 (BD:GK1.5), CD106 (Biolegend:429-MVCAM.A), EpCAM (Biolegend:G8.8), CD44 (Biolegend:IM7), Sca1 (Biolegend:D7). For IFN-γ, cells were pre-stimulated with PMA (20 ng/mL), Ionomycin (Sigma, 1 μg/mL) and Golgi stop (BD, 0.8 μL/106 cells) for 4 hr. Samples were subsequently run using BD FACS LSRII or sorted using BD FACS ARIA. Data were analyzed using FlowJo.
Cd106
Manufactured by BioLegend
Sourced in United States
CD106, also known as VCAM-1, is a cell adhesion molecule that mediates the adhesion of lymphocytes, eosinophils, and monocytes to vascular endothelium. It is expressed on activated endothelial cells and plays a role in cell-cell interactions.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BioLegend (3 mentions)
Sca 1, by BioLegend (1 mentions)
Facs lsr 2, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Cd163, by BioLegend (1 mentions)
Cd271, by BioLegend (1 mentions)
Pd l1, by BioLegend (1 mentions)
I a 1 e, by BD (1 mentions)
Epcam, by BioLegend (1 mentions)
F4 80, by BioLegend (1 mentions)
Ifn γ, by BioLegend (1 mentions)
Cd206, by BioLegend (1 mentions)
S3 cell sorter, by Bio-Rad (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
8 protocols using cd106
1
Multi-parameter Flow Cytometry Profiling
2
Rat BMSC Immunophenotyping by Flow Cytometry
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Rat BMSCs at passage 1 were detached from the cell culture flask by Accutase (Innovative Cell Technologies, San Diego, CA, United States). After washing with phosphate-buffered saline (PBS), the cells were stained with antibodies against CD45 (AbD Serotec, Raleigh, NC, United States), CD73 (BD Biosciences, San Jose, CA, United States), CD90 (BioLegend, San Diego, CA, United States), and CD106 (BioLegend, San Diego, CA, United States). The stained cells were analyzed by flow cytometry with an S3 Cell Sorter (Bio-Rad, Hercules, CA, United States).
3
Isolation and Characterization of Rat Adipose Stem Cells
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The inguinal adipose tissues were harvested from 4-week-old SD rats (purchased from Guangdong Medical Experimental Animal Center) under sterile conditions; type I collagenase (Sigma) was then added for 45-min agitating digestion at 37°C; the mixture was then repetitively centrifuged, and after discarding the supernatant, the cellular pellet (primary cells, P0) was resuspended in DMEM/F12 (Hyclone company) with 10% fetal bovine serum (Gibco) for consecutive passage and purification. The P3 cells were then detected in the surface antigens (CD29, CD45, CD44, and CD106, Biolegend) using FACScan flow cytometer (BD FASCanto™, USA).
4
Comprehensive Immune Cell Profiling
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Cellular phenotypic analysis was carried out by direct immunofluorescence with the following antibodies: CD11b M1/70 (eBioscience), Ly6g 1A8 (eBioscience) F4/80 BM8 (Biolegend), MHCII 2G9 (Biosciences), NK1.1 S17016D (Biolegend), CD3 145-2C11 (eBioscience), CD31 390 (Biolegend), αSMA (α smooth muscle actin) 1A4 (eBioscience), CD62-E REA369 (Miltenyi Biotec), CD62P RMP-1 (Biolegend), CD54 (Biolegend), CD106 (Biolegend), CD192 (CCR2) REA538 (Miltenyi Biotec)., PSGL-1 34 RA10 (Thermofisher), CD44 IM7.8.1 (Miltenyi Biotec), CD11a 121.7 (Thermofisher), CD49d 9C10 MFR4.B (Biolegend). All incubations were done in the presence of 50 μg/ml mouse IgG or human IgG. The same isotype control antibody was always included as a negative control, and dead cells were excluded by Annexin V staining (Sigma, USA). Flow cytometry was performed with a 10 color Gallios device (Beckman Coulter, USA), cells were counted using Flow-Count fluorospheres (Beckman Coulter, USA), following the manufacturer’s instructions, and data were analyzed using Flowjo software (Tree Star, Inc, USA).
5
Immunophenotypic Characterization of Cells
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Surface markers were stained by incubating cells with each corresponding antibody for 30 min in dark according to the product instructions. Each test included an isotype-matched negative control. Cells were then washed twice with PBS containing 1% FBS and resuspended in 0.5 ml PBS. Flow cytometry was performed on a FACScan flow cytometer (Beckman Coulter, CA). Mouse monoclonal antibodies were obtained from the sources indicated: CD73, CD29, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 (eBioscience, San Diego, CA); CD90, CD105, CD106 and CD45 (Biolegend, San Diego, CA); CD34 (BD Pharmingen, San Diego, CA); CD133 (Miltenyi Biotech). Primary antibodies were diluted to the concentration recommended by the supplier (generally 1 μg/ml).
6
Characterization of Mesenchymal Stem Cells
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When the stem cells were passaged to the fifth generation, the cell phenotype was identified by flow cytometry. After trypsinization, the cells were centrifuged at 1000 rpm for 10 min and washed twice with PBS. After resuspension in PBS, the cells were centrifuged at 2000 rpm for 6 min, and the supernatant was discarded. One microliter of antibody was added to each tube, and was incubated for 30 min at 4 °C in the dark. Then, 1 mL PBS was added to each tube and centrifuged at 2000 rpm for 6 min. This step was repeated twice. Finally, the cells were resuspended in 0.5 mL PBS and analyzed using flow cytometry. The antibodies included positive markers (CD29 (catalog #102207, Biolegend, San Diego, CA, USA), CD90 (catalog #202503, Biolegend, San Diego, CA, USA), CD106 (catalog #200403, Biolegend, USA), and negative markers, CD11b (catalog #201807, Biolegend, San Diego, CA, USA), and CD45 (catalog #202207, Biolegend, San Diego, CA, USA). According to FlowJo software (7.6.5, Ashland, Wilmington, DE, USA) analysis, CD29+, CD90+, and CD106+ cells were designated as MSCs, and CD45+ and CD11b+ cells were designated as hematopoietic stem cells.
7
Characterization of BM-MSC Surface Markers
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Cell surface markers of BM-MSCs were quantified by fluorescence-activated cell sorting (FACS) analysis of a homogenous population of fibroblast-like cells obtained from passages 2-4. For direct labeling, a total of 2 Â 10 5 cells were incubated with fluorescein isothiocyanate-conjugated monoclonal rat antimouse antibodies positive to cluster of differentiation 44 (CD44), CD90 (BD Biosciences Pharmingen, California, USA), CD73, CD105 (Santa Cruz Biotechnology, California, USA), and CD106 (Biolegend, San Diego, California, USA) and negative to CD31, CD45 (BD Biosciences Pharmingen) and CD34 (Santa Cruz Biotechnology) for 40 min on ice in darkness. Unstained cells were used as negative control. The analysis was then carried out by using a FACSCalibur cytometer (Becton and Dickinson Co., Franklin Lakes, New Jersey, USA) and CellQuest software (Becton and Dickinson Co.).
8
Bone Marrow Cells for Sepsis Treatment
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The marrow was harvested by inserting a syringe needle (27-gauge) into one end of the bone and flushing with Dulbecco's Modified Eagle's Medium (DMEM; Gibco). The bone marrow cells were filtered through a 70-mm nylon mesh filter (BD, Falcon) and separated under Ficoll gradient to obtain mononuclear cells. These cells were injected intravenously 1 h after in CLP-or LPS-treated mice. A small aliquot of the mononuclear cells was used for immunophenotipic characterization of the injected cell population. Cell characterization was performed by flow cytometry using antibodies CD45, CD34, CD3, CD11b, cKIT, ScaI, CD150, CD29, Ter119, CD106, all from Biolegend.
In vitro studies RAW264.7 cells were seeded at 5 × 10 5 cells per well and cocultivated with bone marrow cells (5 × 10 5 cells) in the presence or not of LPS (1 μg/mL, E. coli 055:B5, Sigma) for 24 h, in 24 wells plates and also in transwells. NS398 (1 μM) was added in trans-well coculture experiments 30 min before LPS treatment to inhibit COX 2 activity.
In vitro studies RAW264.7 cells were seeded at 5 × 10 5 cells per well and cocultivated with bone marrow cells (5 × 10 5 cells) in the presence or not of LPS (1 μg/mL, E. coli 055:B5, Sigma) for 24 h, in 24 wells plates and also in transwells. NS398 (1 μM) was added in trans-well coculture experiments 30 min before LPS treatment to inhibit COX 2 activity.
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