The C-EVs were first labelled with FITC-CD9 according to the manufacturer’s instructions (BD Biosciences, USA). Briefly, 200 μL of the cell-labelling solution was added to 500 μL of an EVs suspension (1.77E+10 Particles/mL) and incubated at 37 °C for 30 min. Subsequently, the mixture was washed by PBS twice to remove the free dye, and then the sediment was suspended with 500 μL of PBS. The HUCMSCs were incubated with labelled C-EVs at 37 °C for 12 h. The HUCMSCs were then fixed with 4% paraformaldehyde at room temperature for 20 min after washing with PBS. Next, cells were dyed with phalloidin and DAPI. Confocal images were sequentially acquired by confocal microscopy (TCS SP8, Leica, Germany).
Fitc cd9
Manufactured by BD
Sourced in United States
FITC-CD9 is a fluorescence-labeled antibody that binds to the CD9 cell surface antigen. CD9 is a member of the tetraspanin family of proteins and is expressed on various cell types, including hematopoietic cells, endothelial cells, and some tumor cells. The FITC (Fluorescein Isothiocyanate) label allows for the detection and identification of CD9-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Tcs sp8, by Leica (4 mentions)
Cd63 fitc, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Actn2 antibody, by Merck Group (1 mentions)
Fortessa analyser, by BD (1 mentions)
Influx cell sorter, by BD (1 mentions)
Pdgfra, by BD (1 mentions)
Epcam pe, by BioLegend (1 mentions)
Goat serum, by Merck Group (1 mentions)
Facsdiva, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 450, by BioLegend (1 mentions)
Cd135 pe, by BD (1 mentions)
Cd127 apc, by BD (1 mentions)
Cd25 bv605, by BioLegend (1 mentions)
Lineage fitc, by BD (1 mentions)
Cd38 bv711, by BD (1 mentions)
Cd34 bv421, by BioLegend (1 mentions)
Cd16 32 percpcy5, by BD (1 mentions)
8 protocols using fitc cd9
1
Visualizing EVs Uptake by HUCMSCs
2
Characterization of Small Extracellular Vesicles
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The sEVs precipitate was resuspended in a trace amount of fresh PBS (200 µL). This suspension was used for NTA, TEM, and flow cytometry. The 50 µL of sEVs stock solution was diluted to 1 mL and the particle size was determined using NTA (Malvern, Malvern City, UK). For TEM,10 µL of the sample was fixed overnight at 4 °C with 2.5% glutaraldehyde solution before being placed onto a copper grid and incubated for 5 min at 25 °C. Next, the copper grid was stained with three drops of saturated uranium dioxide acetate solution for 1 min and then washed with three drops of ddH2O for 5 min at 25 °C. This washing process was repeated once. After drying at 25 °C, the samples were imaged using a TEM (FEI, Hillsboro, Oregon, USA). For flow cytometry, the sEVs stock solution was diluted to 60 µL and by adding 20 µL of FITC-CD9 and FITC-CD63 (BD, Franklin Lakes, NJ, USA) antibodies before being incubated at 25 °C for 40 min. After the addition of 1 mL of pre-cooled PBS, the excess antibody was washed by centrifugation at 100,000 × g for 70 min and the washing step was repeated once more. Samples were resuspended with 100 µL of pre-cooled 1× PBS and detected using flow cytometry (BD).
3
Labeling and Imaging of C-EVs Uptake
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The C-EVs were rst labelled with FITC-CD9 according to the manufacturer's instructions (BD Biosciences, USA). Brie y, 200 μL of the cell-labelling solution was added to 500 μL of an EVs suspension (1.77E + 10 Particles/mL) and incubated at 37°C for 30 minutes. Subsequently, the mixture was washed by PBS twice to remove the free dye, and then the sediment was suspended with 500 μL of PBS. The HUCMSCs were incubated with labelled C-EVs at 37°C for 12 hours. The HUCMSCs were then xed with 4% paraformaldehyde at room temperature for 20 minutes after washing with PBS. Next, cells were dyed with phalloidin and DAPI. Confocal images were sequentially acquired by confocal microscopy (TCS SP8, Leica, Germany).
4
Labeling and Imaging of C-EVs Uptake
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The C-EVs were rst labelled with FITC-CD9 according to the manufacturer's instructions (BD Biosciences, USA). Brie y, 200 μL of the cell-labelling solution was added to 500 μL of an EVs suspension (1.77E + 10 Particles/mL) and incubated at 37°C for 30 minutes. Subsequently, the mixture was washed by PBS twice to remove the free dye, and then the sediment was suspended with 500 μL of PBS. The HUCMSCs were incubated with labelled C-EVs at 37°C for 12 hours. The HUCMSCs were then xed with 4% paraformaldehyde at room temperature for 20 minutes after washing with PBS. Next, cells were dyed with phalloidin and DAPI. Confocal images were sequentially acquired by confocal microscopy (TCS SP8, Leica, Germany).
5
Chondrocyte-Derived Exosome Labeling and Uptake
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Chondrocytes-derived exosomes(C-EXO) were first labeled with FITC-CD9 according to the manufacturer's instruction (BD Biosciences, USA). Briefly, 200 µL of the cell-labeling solution was added to 500 µL of exosomes suspension and incubated at 37 °C for 30 minutes. Subsequently, the mixture was washed by PBS twice to remove the free dye, and then the sediment was suspended with 500µL of PBS. The HUCMSCs were incubated with labeled C-EXO at 37 °C for 12 hours. The HUCMSCs were then fixed with 4% paraformaldehyde at room temperature for 20 minutes after washing with PBS. Next, cells were dyed with phalloidin and DAPI. Confocal images were sequentially acquired by confocal microscopy (TCS SP8, Leica, Germany).
6
Multiparametric Flow Cytometry for Pluripotency and Lineage Markers
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Flow cytometry analysis and sorting of lives cells was performed for GFP, VCAM1 (diluted 1:100, biotin conjugated Abcam ab7224) detected with APC-Streptavidin conjugated secondary (1:100, Biolegend), and PDGFRA (BD Biosciences, 556001) detected with PE/Cy7 conjugated secondary (Biolegend, 405315), as described previously16 (link),18 (link),64 (link). Pluripotency markers used were ECAD (ThermoFisher Scientific, MA1-10192) detected with APC conjugated secondary (1 in 100), EpCAM-PE (Biolegend, 324205, diluted 1:100), CD9-FITC (BD Biosciences, 341646, diluted 1:100) and SSEA4-APC (Biolegend, 330418, diluted 1:100) were detected as For intracellular flow cytometry, cells were harvested with TrypLE Select, fixed in 4% paraformaldehyde for 15 min at room temperature, blocked and permeablised in block buffer consisting of 1 × Perm/Wash Buffer (BD) and 4% goat serum (Sigma) for 15 min at 4 °C. Cells were then incubated with ACTN2 antibody (Sigma, A7811, diluted 1:100) for 1 h at 4 °C and then Alexa Fluor 647 conjugated secondary (ThermoFisher Scientific, A-21235, diluted 1:1000) for 1 h at 4 °C. Collection of flow cytometric data was performed using BD Fortessa™ analyser and analyzed with FlowLogic software (Inivai Scientific). Cell sorting was done using FACS Diva™ and BD Influx™ cell sorters (BD Biosciences).
7
Comprehensive Immune Cell Profiling
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A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.
8
Characterization of Eosinophils in Systemic Sclerosis
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To ensure that the CD16 low CD9 high granulocytes were eosinophils, five additional SSc patients were analysed for the expression of the eosinophil markers CD193 (CCR-3) and Siglec-8 on a FACSCanto II using the DIVA 6.3 software (Becton Dickinson, BD, New York, NY, USA) and similar gating, Supplementary Figure . The following antibodies were used in this analysis: CD9-FITC, Siglec 8-PE, CD14-PerCP-Cy5.5, CD16-APC-H7, CD193-BD Horizon v500 all from BD bioscience. Two of these samples were subjected to cell sorting on a FACSAria II instrument and the CD16 low /CD9 high population were sorted directly on glass slides and studied under microscope.
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