Cd105 fitc

Manufactured by Bio-Rad

Sourced in United States, United Kingdom

CD105-FITC is a fluorescently labeled antibody that binds to the CD105 antigen. CD105 is a cell surface glycoprotein that is expressed on endothelial cells. This product can be used for the detection and analysis of CD105-positive cells in flow cytometry applications.

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FITC mouse anti-human CD105 CD105

8 protocols using cd105 fitc

Flow Cytometry Analysis of Cell-Derived Particles

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Flow cytometry for assessment of cell surface and MP expression from cancer cells lines and HUVEC was performed on a FACSCalibur (Becton Dickinson, Oxford, UK). For assessment of cell free media was obtained by centrifugation at 400g for 5 minutes to pellet cells. 25µl of this media was then incubated with 5µl of antibody (CD31:FITC, CD54:FITC, CD105:FITC, CD106:FITC or CD142:FITC) (Bio-Rad, Hemel Hempstead, UK) at room temperature in the dark for 30 minutes, when counting beads (Accucheck, Thermo-Fisher) and 150µl filtered PBS were added prior to assessment by flow cytometry using ISTH guidelines for enumeration of MP.
For assessment of cancer or HUVEC cells, 5µg of antihuman antibodies (CD31:FITC, CD54:FITC, CD105:FITC, CD106:FITC or CD142:FITC, Bio-Rad, Hemel Hempstead, UK) or an isotype matched negative control (IgG1:FITC, Bio-Rad) was then added to 50µl of cells (2x10 5 ) and incubated for 30 minutes, at room temperature in the dark. Cells were then washed in PBS and centrifuged. The pellet was then resuspended in 200µl filtered PBS and assessed by flow cytometry.

Faulkner L.G., Alqarni S., Maraveyas A, & Madden L.A. (2020). Isolated tumour microparticles induce endothelial microparticle release in vitro. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 31(1).

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Immunophenotyping of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

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hUCB-MSCs (from P2 to P8) were harvested, washed, and resuspended in PBS-sodium azide buffer (0.1%) at a density of 10⁶cells/ml. Cell suspensions were incubated with monoclonal antibodies for 25 min at 4 °C in a dark room. The following mouse monoclonal anti-human antibodies were used: CD14-Fluorescein isothiocyanate (FITC), CD34-allophycocyanin (APC), CD45-peridinin chlorophyll protein complex (PerCP), CD64-phycoerythrin (PE), CD73-PE, CD146-PE, CD166-PE, HLA-DR-FITC (BD Pharmigen), CD29-FITC, CD44-PE, CD90-PE (IOTest) and CD105-FITC (ABd Serotec). The respective mouse isotype antibodies served as controls. Subsequently, the cells were washed and resuspended in 300 μl PBS-azide. Data acquisition was performed in a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo Software (TreeStar).

Gómez-Leduc T., Hervieu M., Legendre F., Bouyoucef M., Gruchy N., Poulain L., de Vienne C., Herlicoviez M., Demoor M, & Galéra P. (2016). Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering. Scientific Reports, 6, 32786.

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Equine MSC Surface Marker Characterization

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Passage 3 eDPCs (2 × 10⁵ cells) were resuspended in 500 µl of FACS buffer (PBS containing 1% sodium azide and 0.5% BSA, pH 7.2).
The cells were incubated with various antibodies, including CD11a/CD18 (Clone CZ3.2, 117, 2E11, B10; Gifts from Dr. Douglas Antczak, Cornell University, Ithaca,
NY, U.S.A.), CD34-FITC (Clone CA 581/CD34; BD Biosciences, San Jose, CA, U.S.A.), CD44 (Clone CVS18; AbD Serotec, Raleigh, NC, U.S.A.), CD45-FITC (Clone 2D1;
BD), CD90-FITC (Clone 5E10; BD), CD105-FITC (Clone SN6; Serotec), MHC Class I (Clone CZ3, 117, 1B12, C11; Gifts) and MHC Class II (Clone CZ11, 130, 8E8, D9;
Gifts) at 4°C for 30 min. All clones have been previously used in equine MSC experiments [15 (link),16 (link),17 (link),18 (link)]. The cells were washed twice with FACS buffer and
resuspended in 500 µl of FACS buffer. The cells incubated with the CD11a/CD18, CD44 and MHC class I and II antibodies were incubated with
anti-mouse IgG secondary antibodies labeled with FITC (Rockland, Gilbertsville, PA, U.S.A.) at 4°C for 30 min. Nonspecific FITC mouse immunoglobulin G1κ was
used as a negative control. Cells were then washed twice with FACS buffer and resuspended in 500 µl of FACS buffer. Cell fluorescence was
evaluated by flow cytometry with a FACSCalibur instrument (BD Biosciences). Data were analyzed using CellQuest Pro software (BD Biosciences).

ISHIKAWA S., HORINOUCHI C., MURATA D., MATSUZAKI S., MISUMI K., IWAMOTO Y., KOROSUE K, & HOBO S. (2016). Isolation and characterization of equine dental pulp stem cells derived from Thoroughbred wolf teeth. The Journal of Veterinary Medical Science, 79(1), 47-51.

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Phenotypic Characterization of BM-MSCs

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BM-MSCs (from 8 donors between P3 and P7) were harvested, washed, and resuspended in PBS-sodium azide buffer (0.1%) at a density of 10 × 10⁶ cells/ml. Cell suspensions (0.5 × 10⁶ cells/50 µl) were incubated with 3 to 5 µl of monoclonal antibodies for 25 min at 4 °C in the dark. The following anti-human antibodies were used: CD14-fluorescein isothiocyanate (FITC; BD Biosciences), CD34-allophycocyanin (APC; BD Biosciences), CD45-peridinin chlorophyll protein complex (PerCP; BD Biosciences), CD64-phycoerythrin (PE; BD Biosciences), CD73-PE (BD Pharmingen), CD146-PE (BD Pharmingen), HLA-DR-FITC (BD Biosciences), CD29-FITC (Beckman Coulter), CD44-PE (Beckman Coulter), CD90-PE (Beckman Coulter), and CD105-FITC (ABd Serotec). The respective mouse isotype antibodies served as controls. The cells were then washed and resuspended in 300 μL PBS-azide. Acquisitions were performed in a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed using FlowJo Software (TreeStar).

Legendre F., Ollitrault D., Gomez-Leduc T., Bouyoucef M., Hervieu M., Gruchy N., Mallein-Gerin F., Leclercq S., Demoor M, & Galéra P. (2017). Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-β1, hypoxia, and COL1A1/HtrA1 siRNAs. Scientific Reports, 7, 3406.

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Multiparametric Immunophenotyping of ASP-ADSCs

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ASP-and GEMS-ADSCs were evaluated for the expression of mesenchymal, hematopoietic, endothelial, and immunological markers: anti-human CD14-PE, CD31-PE, CD34-PE, CD45-FITC, CD73-PE, HLA-DR-FITC (BD Pharmingen, San Jose, CA), and CD105-FITC (AbDSerotec, Raleigh, NC). Briefly, 5 • 10 4 cells/tube were stained with fluorochrome-conjugated monoclonal antibodies and incubated for 15 min at RT in the dark. Samples were centrifuged at 300 g for 5 min, washed twice with D-PBS and then fixed with 4% paraformaldehyde (PFA). Isotypematched mouse immunoglobulins were used as control. At least 5 • 10 3 events were acquired for each sample. Nonviable cells were excluded by physical gating. For data acquisition, a FACS Canto II (BD Pharmingen) with DIVA software was used, whereas the analysis of the data was performed using the Cell Quest software (BD Pharmingen).

Marfia G., Navone S.E., Hadi L.A., Paroni M., Berno V., Beretta M., Gualtierotti R., Ingegnoli F., Levi V., Miozzo M., Geginat J., Fassina L., Rampini P., Tremolada C., Riboni L, & Campanella R. (2016). The Adipose Mesenchymal Stem Cell Secretome Inhibits Inflammatory Responses of Microglia: Evidence for an Involvement of Sphingosine-1-Phosphate Signalling. Stem cells and development, 25(14).

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Immunophenotypic Characterization of MSCs

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The cells were incubated with antibodies against NG2 PE (Beckman Coulter), CD45 PC5 (Beckman Coulter, Marsillia, France), CD73 PE (Becton-Dickinson, Bioscience Pharmingen, San Diego, CA, USA), and CD105 FITC (Serotec, Oxford, UK). Fluorescence histograms were obtained by recording 20,000 cells/sample at a flow rate of approximately 200 cell events/s. Experiments were performed using Coulter Epics XL-MCL and flow cytometric data were analyzed using the EXPO 32 ADC software (Beckman Coulter Inc, Miami, FL, USA) [22] . Flow cytometric data analysis demonstrated that there were significant expressions of MSC-specific antigens (CD105, CD73, and NG2) and the absence of hematopoietic marker antigen (CD45) [23] .

Işık S., Karaman M., Adan A., Kıray M., Bağrıyanık H.A., Sözmen Ş.Ç., Kozanoğlu İ., Karaman Ö., Baran Y, & Uzuner N. (2017). Intraperitoneal mesenchymal stem cell administration ameliorates allergic rhinitis in the murine model. European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery, 274(1).

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Flow Cytometry Analysis of MSC Markers

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The characteristic surface markers of MSCs [34 ] were examined by FACS analysis. Cells were washed with phosphate-buffered saline containing 0.5% bovine serum albumin and 1 mM EDTA. The cells were then incubated with the antibodies to CD34-APC, CD45-APC, HLA-DR-FITC (1:40; Thermo Fisher Scientific Inc., Waltham, MA, USA), CD90-APC (1:100; BD Biosciences, San Jose, CA, USA), and CD105-FITC (1:20; Bio-Rad Laboratories, Inc., Irvine, CA, USA) for 30 min at 4 °C. IgG1-FITC (1:40; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), IgG1-APC, and IgG2a-FITC (1:40; Thermo Fisher Scientific, Inc.) were used as isotype-matched negative controls. Propidium iodide was used to remove nonviable cells. Moreover, cell surface marker expression was analyzed by Gallios (Beckman Coulter, Brea, CA, USA). Furthermore, data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Fukushima K., Itaba N., Kono Y., Okazaki S., Enokida S., Kuranobu N., Murakami J., Enokida M., Nagashima H., Kanzaki S., Namba N, & Shiota G. (2021). Secreted matrix metalloproteinase-14 is a predictor for antifibrotic effect of IC-2-engineered mesenchymal stem cell sheets on liver fibrosis in mice. Regenerative Therapy, 18, 292-301.

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Mesenchymal Stem Cell Characterization

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After each recovery period, cells were detached with TripLE Express (Thermo Scientific, USA), centrifuged and resuspended in cold PBS with 1% (v/v) FBS. Then, fluorescence-labeled mouse anti-human antibodies CD105-FITC (Bio-Rad, Netherlands), CD73-PE (BD Biosciences, Germany) and CD90-APC (BD Biosciences, Germany), were incubated 20 min at room temperature, using manufacturers recommended concentrations. Cells were washed with PBS, centrifuged, resuspended and analyzed in a FACSCalibur flow cytometer (BD Biosciences, USA). Cell viability was also assessed after labeling cells with 7-AAD (BioLegend, USA). Results were analyzed using Cyflogic software (1.2.1, CyFlo Ltd, Finland).

Freitas-Ribeiro S., Carvalho A.F., Costa M., Cerqueira M.T., Marques A.P., Reis R.L, & Pirraco R.P. (2019). Strategies for the hypothermic preservation of cell sheets of human adipose stem cells. PLoS ONE, 14(10), e0222597.

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