Cd105 fitc

Manufactured by Miltenyi Biotec

Sourced in Germany

CD105-FITC is a fluorochrome-conjugated monoclonal antibody that specifically binds to the CD105 (Endoglin) antigen. CD105 is a transmembrane glycoprotein that is primarily expressed on the surface of endothelial cells. The FITC (Fluorescein Isothiocyanate) fluorochrome enables the detection and analysis of CD105-positive cells using flow cytometry and other fluorescence-based techniques.

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CD105 (27 citations)

10 protocols using cd105 fitc

Characterization of Mesenchymal Stem Cells

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Selected early and senescent passage cells were defrosted from frozen vials in a water bath at 37 °C. The cells were counted to be of at least

10^{5}

cells per tube. The cell pellet was re-suspended in 200

μ l

of blocking buffer (0.5% BSA—bovine serum albumin, 2% FBS in 1× PBS) and incubated for 15 min at room temperature. FACS buffer (0.5% BSA, 0.05% Sodium Azide in 1× PBS) of 200

μ l

were added to the suspension and the solution was split into tubes with 50

μ l

each. Antibodies against MSC positive markers (Miltenyi Biotec): CD73-PE (Clone AD2), CD90-PerCP-Vio700 (Clone REA897), CD105-FITC (Clone 43A4E1) and negative markers (Viogreen): CD14 (Clone REA599), CD19 (Clone LT19), CD34 (Clone AC136), CD45 (Clone REA747), HLA-DR (Clone REA805) were added and solutions were incubated for 15 min at 4 °C in the dark. FACS buffer of 500

μ l

were added to each tube to wash off non-binding antibodies. Tubes of stained and unstained cells were spun down at 400 rcf for 5 min, re-suspended in 500

μ l

FACS buffer and the data were acquired by Attune Acoustic Focusing Flow Cytometer (Applied Biosystems). The FlowJo software (version 10.7) (http://www.flowjo.com/solutions/flowjo/downloads) was used for data analysis with debris excluded by gates, and the percentage of cell expressing these surface markers were also recorded.

Zhai W., Tan J., Russell T., Chen S., McGonagle D., Win Naing M., Yong D, & Jones E. (2021). Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods. Scientific Reports, 11, 1054.

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Characterization of Mesenchymal Stem Cells

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Cells were harvested by trypsinization (0.05% (w/v); Life Technologies, Cat. #15400-054) for 2 min, and the cell pellet was resuspended in PBS to a titer of 10⁶/100 μL. The cell suspension was incubated with the following antibodies respectively. CD34-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat. #130-098-142), CD45-PE (Miltenyi Biotec, Cat. #130-080-201), CD73-PE (Miltenyi Biotec, Cat. #130-112-060), CD90-FITC (Miltenyi Biotec, Cat. #130-095-403) and CD105-FITC (Miltenyi Biotec, Cat. #130-112-327). About 2 μL of each antibody (1:10 diluted according to the datasheet of the antibodies) per tube was added and incubated for 10 min in darkness at 2–8 °C. Cells were washed with PBS completely and resuspended in 1% (w/v) paraformaldehyde. Samples were run on a FC500MPL flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed by FlowJo vX.0.7 software (FlowJo LLC, Ashland, OR, USA).

Wang Y., Yu D., Liu Z., Zhou F., Dai J., Wu B., Zhou J., Heng B.C., Zou X.H., Ouyang H, & Liu H. (2017). Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix. Stem Cell Research & Therapy, 8, 189.

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Isolation and Characterization of CD146+ MSCs

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CD146⁺/CD45^-/HLA-DR^--MSC were isolated from noncultured BM-MNC using multicolor FACS using standard protocols (ARIA II; BD Biosciences). Immunophenotype of CFU-F–derived MSC and CD146⁺/CD45^-/HLA-DR^--MSC was analyzed using flow cytometry (LSRFortessa, FACSDiva software; BD Biosciences) according to a standardized protocol. The evaluated antigens were selected according to the International Society for Cellular Therapy consensus.⁶ (link) For FACS and flow cytometric analyses the following mouse anti-human antibodies were used: CD90-PE, CD105-FITC, CD146-PE, CD45-FITC, HLA-DR-FITC, CD34-FITC, CD73-PE, CD14-PE, and CD19-FITC (all Miltenyi Biotec).

Katzerke C., Schaffrath J., Lützkendorf J., Janssen M., Merbach A.K., Nerger K., Binder M., Baum C., Lauer K., Rohde C., Willscher E., Müller-Tidow C, & Müller L.P. (2023). Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome. Blood Advances, 7(12), 2811-2824.

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Comprehensive Immunophenotyping of WT and TNFR2 KO MSCs

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A total of 10⁵ WT and TNFR2 KO-MSCs were seeded in 96-well round-bottom plates (Falcon). Thereafter, they were immunostained using the following Abs: CD44-PE-Vio770, CD105-FITC, Sca1-APC, CD73-PE, CD90-Biotin or PE, anti-biotin-PE or VIOBLUE, CD45-VIOBLUE, CD34-Biotin or FITC (Miltenyi), and streptavidin-PeCY5/CY7 (eBioscience). We used isotypes Abs (Miltenyi) and unstained MSCs as controls for our experiments.
For F-actin staining, we have used F-actin Staining Kit, Green Fluorescence, Cytopainter (Abcam) according to supplier’s protocol. Briefly, we have seeded 10⁵ WT and TNFR2 KO MSCs in 96-well plates, treated them with 100 μl/well of Green Fluorescent Phalloidin Conjugate solution, and stained them in room temperature for 30 min. We then washed the cells with PBS 1× and analyzed in FITC channel using LSRFORTESSA flow cytometer (BD Biosciences). Data were then analyzed using FlowJo software v10 (FlowJo, LLC).

Beldi G., Bahiraii S., Lezin C., Nouri Barkestani M., Abdelgawad M.E., Uzan G, & Naserian S. (2020). TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties. Frontiers in Cell and Developmental Biology, 8, 596831.

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Flow Cytometry Analysis of MSC Phenotype

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Surface expression of culture‐expanded MSCs was assessed using the ISCT criteria of cultured MSCs.³³ Briefly, after trypsinization, cells were pelleted by centrifugation at 500 g for 10 minutes, stained with relevant antibodies (all from Miltenyi Biotec) in the dark at 4°C for 15 minutes (positive markers: CD73‐PE (Clone AD2,), CD90‐PerCP‐Vio700 (Clone REA897), CD105‐FITC (Clone 43A4E1); negative markers (all VioGreen): CD14 (Clone REA599), CD19 (Clone LT19) CD34 (Clone AC136), CD45 (Clone REA747), HLA‐DR (Clone REA805)). Data acquisition was conducted using the Attune 2 Laser System (Invitrogen) and analysed using the Attune Cytometric Software (v2.1.0). Gates were applied to discount debris and then to measure the percentage of cells expressing these surface markers.

Jones W.G., El‐Jawhari J.J., Brockett C.L., Koria L., Ktistakis I, & Jones E. (2020). Multipotential stromal cells in the talus and distal tibia in ankle osteoarthritis – Presence, potency and relationships to subchondral bone changes. Journal of Cellular and Molecular Medicine, 25(1), 259-271.

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Characterization of Pluripotent Stem Cells

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The cells were detached, dissociated into single cells using dispase (Sigma-Aldrich), and resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline PBS, 1% BSA; 5 × 10⁵ cells/100 μl FACS buffer). For cell-surface staining the cells were stained with primary antibodies CD73-PE (130-095-182, Miltenyi), CD105-FITC (130-094-926, Miltenyi), CD90- PE-VIO770 (130-099-295, Miltenyi), CD45-PE (130-098-141, Miltenyi), CD34-FITC (130-098-139, Miltenyi), CD24-FITC (130-098-861, Miltenyi), SSEA4-PE (FAB1435P, R&D), and TRA1-60-PE (09-0009, Stemgent) for 1 hour at 4 °C. For intracellular FACS staining, i.e. OCT4-A (130-105-606, Miltenyi) and OCT4-B (IC1759P, R&D), the cells were washed once in FACS buffer, fixed for 10 min in 0.01% paraformaldehyde (PFA), washed twice with PBS, resuspended in permeabilization buffer (PBS, 1% Triton) and stained as describe above. Cells were then washed twice with PBS, resuspended in FACS buffer and analysed by FACScalibur flow cytometry (CyAn ADP, Beckman Coulter). Collected data were analyzed with the Flowjo v.10 software package (Treestar, Ashland, OR, USA). Negative control was immunoglobulin G (IgG) primary antibody-specific isotypes.

Hau K.L., Ranzoni A.M., Vlahova F., Hawkins K., De Coppi P., David A.L, & Guillot P.V. (2017). TGFβ-induced osteogenic potential of human amniotic fluid stem cells via CD73-generated adenosine production. Scientific Reports, 7, 6601.

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Immunophenotypic analysis of stem cells

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Immunophenotypic analyses with flow cytometry were performed according to manufacturer’s recommendations. Briefly, 1 × 10⁶ cells per sample (Passage 3–4) were centrifuged at 300× g and resuspended in 100 µl of buffer. Afterwards, 10 µl of the respective antibodies, CD44-VioBlue, CD73-APC, CD90-PE, CD105-FITC, CD146-PE-Vio770 and CD271-APC-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany) and a cocktail of CD14/CD20/CD34/CD45-PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), was added to cell suspension and incubated for 10 min in the dark in the refrigerator. Then, cells were washed with 2 mL of buffer and centrifuged. Supernatant was aspirated, and the final sediment was resuspended in buffer for flow cytometry analysis. Similarly, respective iso-types controls were used to assess background fluorescence and non-specific binding of anti-bodies to cells. All data were acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) and further analyzed by MACS Quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany).

Culenova M., Nicodemou A., Novakova Z.V., Debreova M., Smolinská V., Bernatova S., Ivanisova D., Novotna O., Vasicek J., Varga I., Ziaran S, & Danisovic L. (2021). Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells. International Journal of Molecular Sciences, 22(22), 12503.

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Mesenchymal Stem Cell Characterization

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AFSCs were detached, washed with FACS buffer (PBS supplemented with 1% BSA, Sigma), and stained with primary antibodies CD73-PE, CD90-APC and CD105-FITC (Miltenyi) for 1 hour at 4 °C. Cells were then washed again with the FACS buffer and analysed by FACScalibur flow cytometry (Becton Dickinson).

Ranzoni A.M., Corcelli M., Hau K.L., Kerns J.G., Vanleene M., Shefelbine S., Jones G.N., Moschidou D., Dala-Ali B., Goodship A.E., De Coppi P., Arnett T.R, & Guillot P.V. (2016). Counteracting bone fragility with human amniotic mesenchymal stem cells. Scientific Reports, 6, 39656.

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Isolation and Characterization of Murine Bone Marrow-Derived Mesenchymal Stem Cells

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BM-MSCs were isolated from the femurs and tibias of 6- to 8-week-old C57BL/6 WT mice (Charles River and Envigo) and C57BL/6 TNFR2 KO (B6.129S2-Tnfrsf1b^tm1Mwm/J, The Jackson Laboratory). Mice were housed under pathogen-free conditions. Cells were cultured in 25-cm² flasks in MEMα medium (Gibco) containing low glucose, 1% GlutaMAX, 10% FBS, and 1% penicillin/streptomycin/neomycin (P/S/N) (Gibco). Cells were incubated at 37 °C in a 5% CO₂. Non-adherent cells were removed every 8 h; pure MSCs were obtained after 4–5 weeks. Cells were subcultured prior to confluency. In all experiments, WT and TNFR2 KO MSCs were used in passages 2 to 4.
For the identification of MSCs, 10⁵ cells/well WT and TNFR2-MSCs were seeded in Falcon 96-well round-bottom plates. They were then immunostained with CD44-PeCy7, Sca1-APC, CD105-FITC, CD73-PE, CD45-VIOBLUE, CD34-Biotin or FITC, CD90-Biotin or PE and anti-biotin-PE or VIOBLUE (Miltenyi), and streptavidin-PeCY5/CY7 (eBioscience). Unstained cells and isotypes were used as controls. Flow cytometric analysis was performed using the LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using the FlowJo software v10 (FlowJo-LLC).

Beldi G., Khosravi M., Abdelgawad M.E., Salomon B.L., Uzan G., Haouas H, & Naserian S. (2020). TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function. Stem Cell Research & Therapy, 11, 281.

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Characterization of Mesenchymal Stem Cells by Flow Cytometry

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The isolated ah-BM-MSCs were characterized by means of flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ, USA; Navios Software v1.2) for mesenchymal (CD90, CD73, 105) and hematopoietic (CD34, CD45) markers as previously described [42 (link),44 (link),45 (link)]. Before performing the in vitro assays, single-cell suspensions obtained using culture trypsinization were labeled with fluorochrome-conjugated antibodies: CD73-PE, CD90-APC, CD105-FITC, CD34-APC, and CD45-FITC (Human MSC Phenotyping Cocktail, Miltenyi Biotec, Bergisch Gladbach, Germany) in order to verify the purity of ah-BM-MSCs populations.

López-González I., Hernández-Heredia A.B., Rodríguez-López M.I., Auñón-Calles D., Boudifa M., Gabaldón J.A, & Meseguer-Olmo L. (2023). Evaluation of the In Vitro Antimicrobial Efficacy against Staphylococcus aureus and epidermidis of a Novel 3D-Printed Degradable Drug Delivery System Based on Polycaprolactone/Chitosan/Vancomycin—Preclinical Study. Pharmaceutics, 15(6), 1763.

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