To analyze the expression of specific cell surface proteins on AD-MSCs and BM-MSCs, flow cytometric analysis was performed (n=5 AD-MSCs and n=5 BM-MSCs). The cells from the third passage were trypsinized into single-cell suspensions and labeled with the following anti-mouse antibodies conjugated to fluorochromes: PerCP-Cy5.5-CD11b (0.25 μg/100 μl; BD Biosciences, Bedford, MA, USA), PE-CD14 (0.25 μg/100 μl; BioLegend, CA, USA), PE-CD34 (0.25 μg/100 μl; BioLegend), PE-CD44 (10 μl/100 μl; BioLegend), APC-CD45 (0.25 μg/100 μl; BioLegend), PE-CD49d (0.25 μg/100 μl; BioLegend), PE-CD73 (0.25 μg/100 μl; BioLegend), PE-CD90.2 (0.25 μg/100 μl; BioLegend), PE-CD105 (0.25 μg/100 μl; Abcam, Cambridge, UK), PE-CD106 (0.25 μg/100 μl; BioLegend), APC-CD133 (0.25 μg/100 μl; BioLegend) and PE-Sca-1 (0.5 μg/100 μl; BioLegend). Cells were incubated on ice for 45 minutes with each antibody. Corresponding mouse isotype antibodies were used as controls (1:5; Santa Cruz Biotechnology, TX, USA). The cells were analyzed using the fluorescence-activated cell sorting instrument (FACSCanto II; BD Biosciences) according to the manufacture’s protocol. The percentage of expressed antigen was calculated for 10,000 gated-cell events and the data processed (FACSDiva software; BD Biosciences).
Cd106 pe
Manufactured by BioLegend
Sourced in United States
CD106-PE is a fluorochrome-conjugated antibody that binds to the CD106 (VCAM-1) cell surface antigen. CD106 is an adhesion molecule expressed on the surface of endothelial cells and plays a role in leukocyte-endothelial cell adhesion and migration.
Automatically generated - may contain errors
Lab products found in correlation
Cd90 fitc, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd45 apc, by BioLegend (1 mentions)
Cd90 pe, by BioLegend (1 mentions)
Cd34 pe, by BioLegend (1 mentions)
Cd14 pe, by BioLegend (1 mentions)
Cd73 pe, by BioLegend (1 mentions)
Cd105 pe, by Abcam (1 mentions)
Cd44 pe, by BioLegend (1 mentions)
Percp cy5.5 cd11b, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd45 fitc, by BioLegend (1 mentions)
Cd44 fitc, by BioLegend (1 mentions)
Flow cytometry, by BD (1 mentions)
F4 80 apc bm8, by BioLegend (1 mentions)
Gr1 fitc rb6 8c5, by BioLegend (1 mentions)
Cd44 pe im7, by BioLegend (1 mentions)
Cd3 pe 145 2c11, by BioLegend (1 mentions)
Cd11b fitc m1 70, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd106 pe
1
Cell Surface Protein Expression in MSCs
2
Flow Cytometric Characterization of Tendon-Derived Stem Cells
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To verify the stemness of the cells, TDSCs at passage 2 were incubated with FITC-CD44 (1 : 1000) (BioLegend, San Diego, CA, USA), FITC-CD90 (1 : 8000) (BioLegend), FITC-CD45 (1 : 1000) (BioLegend), and PE-CD106 (1 : 1000) (BioLegend) in the dark at 4°C for 20 minutes. After being washed three times with PBS, TDSCs were analyzed by a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).
3
Phenotypic Characterization of hADSCs
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Flow cytometry (BD Pharmingen, San Diego, CA, USA) was performed on hADSCs at passage 5. The cells were washed three times with phosphate-buffered saline (PBS) and counted under a microscope. Cells were analyzed using conjugated mouse monoclonal antibodies. The cells were washed once in flow wash buffer (1× Dulbecco’s PBS, 0.5% bovine serum albumin and 0.1% sodium azide), resuspended in blocking buffer (wash buffer with 25 μg/mL mouse IgG), and incubated for 10 minutes at room temperature. A total of 100 μL of cell suspension (1 × 105 cells) was added to each tube, and labeled monoclonal antibodies were added for analysis (FITC and PE). PE isotype control was performed for CD13 PE, CD34 PE and CD106 PE. FITC isotype control was performed for CD45 FITC and CD90 FITC. Antibodies were purchased from BD Pharmingen, and CD106 PE was purchased from BioLegend (San Diego, CA, USA). All tubes were incubated and protected from light for 30 minutes. Following incubation, the cells were washed twice with wash buffer and fixed in 200 μL 4% paraformaldehyde. Results were analyzed using Diva analytical software (BD Pharmingen).
4
Flow Cytometry Analysis of Bone Marrow and Spleen Cells
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Bone marrow and spleen cells were stained for flow cytometry as previously described (Mayer et al., 2017 (link)). Gating strategy for BM erythroid cells was described in the Supplementary Figure 1 . Cells were stained with the following antibodies: CD45-FITC (30-F11, eBioscience), CD11b-FITC (M1/70, eBioscience), Gr-1-FITC (RB6-8C5, eBioscience), Ter119-APC (TER119, Biolegend) and CD44-PE (IM7, Biolegend), F4/80-APC (BM8, Biolegend), Gr-1-FITC (RB6-8C5, Biolegend), CD3-PE (145-2C11, Biolegend), Ly6C-PE (HK1.4, Biolegend), CD106-PE (429, Biolegend), CD45R/B220-APC (RA3.682, Biolegend). Cells were analyzed using AccuriC6 flow cytometer and data were analyzed using FlowJo software (v10.7.1).
5
Flow Cytometric Analysis of Mesenchymal Markers in ADSCs
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For analysis of mesenchymal surface markers, ADSCs in passages three to five were trypsinized, washed with staining buffer (BD Pharmingen, BD Biosciences, Heidelberg, Germany), and stained with the following antibodies: CD29-PE, CD34-PE, CD90-FITC (all BD Biosciences), CD31-PE, CD45-PE, and CD106-PE (all Biolegend, London, UK) on ice and in the dark for 20 min. Fluorochrome-conjugated isotype control antibodies (BD Biosciences) were used to determine the level of nonspecific binding. Samples were washed (300× g, 4 °C, 5 min) and resuspended with staining buffer. The cells were analyzed by flow cytometry directly after incubation with 7-aminoactinomycin (7-AAD, Biolegend) for 10 min on ice and protected by light (Cytomics FC 500, Beckmann Coulter, Krefeld, Germany). Positive and negative events were calculated using the CXP™ software (Beckman Coulter) and gated for living cells (negative for 7-AAD).
6
Characterization of ASC Surface Markers
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Characteristic surface markers (cluster of differentiation, CD) of the S-ASCs and V-ASCs from three animals were determined using flow cytometry (FACSCalibur; BD Biosciences, Mississauga, ON, Canada). Briefly, 10 6 live cells at the third to fifth passages were suspended in 100 µl wash flow buffer (WFB). The WFB contains 1× PBS, 2% FBS, 0.05% sodium azide (Sigma-Aldrich). Then the ASCs were incubated with antibodies of interest at 4°C for 30 min. The antibodies used in this study included CD11b-Alexa Fluor 488 (1:200 dilution, 201812 ; Biolegend, San Diego, CA, USA), CD29-Alexa Fluor 488 (1:50 dilution, 102212; Biolegend), CD31-Alexa Fluor 488 (1:10 dilution, MCA1334A488; AbD Serotec, Raleigh, NC, USA), CD45-phycoerythrin (PE) (1:100 dilution, 202207; Biolegend), CD59-fluorescein isothiocyanate (FITC) (1:20 dilution, 550976; BD Biosciences), CD90-FITC (1:200 dilution, 202504; Biolegend) , and CD106-PE (1:20 dilution, 200403; Biolegend) . For assessment of CD73 expression, the ASCs were incubated with primary antibody CD73 (1:50 dilution, 551123; BD Biosciences) at 4°C for 30 min and then washed with PBS three times and stained with second antibody goat anti-mouse IgG-FITC (1:100 dilution, sc-2010; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 1 h. FACS was performed using 20,000 cells per sample.
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