Cd140b

Manufactured by BD

Sourced in United States

CD140b is a cell surface protein that is a member of the platelet-derived growth factor receptor (PDGFR) family. It is commonly used as a marker for the identification and isolation of mesenchymal stem cells and pericytes.

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Lab products found in correlation

Annexin 5, by BD (2 mentions) Cd146, by BD (2 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Hla dr, by BD (1 mentions) Cd105, by BD (1 mentions) Cd29 apc, by BioLegend (1 mentions) Epics altra flow cytometer, by Beckman Coulter (1 mentions) Cd24 pe, by BioLegend (1 mentions) Cd49f fitc, by BioLegend (1 mentions) Cd24 pe, by Thermo Fisher Scientific (1 mentions) Easysep magnet, by STEMCELL (1 mentions) Aldefluor kit, by STEMCELL (1 mentions) Macs running buffer, by Miltenyi Biotec (1 mentions) Cd144, by BD (1 mentions) Facscanto, by BD (1 mentions) H 2kd, by BioLegend (1 mentions) Moflo astrio, by Beckman Coulter (1 mentions) Dcfh da, by Merck Group (1 mentions) Propidium iodide, by Merck Group (1 mentions) Cytoflex, by Beckman Coulter (1 mentions)

7 protocols using cd140b

Mesenchymal Stem Cell Immunophenotyping

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Cells were harvested with 0.05% trypsin-EDTA solution, washed, suspended in PBS supplemented with 2% FBS, incubated with antibodies to phycoerythrin-conjugated antibodies CD 90, CD 105, CD 73, CD, 140b, HLA DR (all from BD Pharmingen), CD 44 (eBioscience, USA) for 30 min in flow cytometry tubes at room temperature in the dark following the manufacturer instructions, and analyzed using CytoFLEX flow cytometer (Beckman Coulter, USA; 561 nm laser).

Zenin V., Ivanova J., Pugovkina N., Shatrova A., Aksenov N., Tyuryaeva I., Kirpichnikova K., Kuneev I., Zhuravlev A., Osyaeva E., Lyublinskaya E., Gazizova I., Guriev N, & Lyublinskaya O. (2022). Resistance to H2O2-induced oxidative stress in human cells of different phenotypes. Redox Biology, 50, 102245.

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Isolation and Characterization of Breast Cancer Stem Cells

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Freshly isolated mouse mammary cells were incubated with biotinylated anti-CD31, CD45, and Ter119 cocktail and the labeled Lin⁺ cells were removed by EasySep magnet (StemCell Technologies). The Lin⁻ cells were incubated with fluorescence-conjugated antibodies, including CD24-PE, CD29-APC, and CD49f-FITC antibodies (all from Biolegend), as described (24 (link), 25 (link)). For human cell lines, CD24-PE and CD44-APC antibodies (eBioscience) were used to fractionate the BCSC-enriched population, as described (26 (link)). Isotype antibodies were used as negative controls. Sorting for BCSCs freshly isolated from human breast cancers was performed with Epics Altra flow cytometer (Beckman Coulter). To deplete non-tumor cells from primary cancer samples, a cocktail of lineage marker antibodies including CD2, CD3, CD10, CD16, CD18, CD31, CD64 and CD140b (PharMingen) were used, while to deplete non-tumor cells from mouse specimens, anti-H2 Kd antibody was used.
The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with a high ALDH enzymatic activity, as described (27 (link)). As negative control, for each sample of cells an aliquot was treated with a specific ALDH inhibitor diethylaminobenzaldehyde (DEAB).

Luo M.L., Gong C., Chen C.H., Lee D.Y., Hu H., Huang P., Yao Y., Guo W., Reinhardt F., Wulf G., Lieberman J., Zhou X.Z., Song E, & Lu K.P. (2014). Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer. Cancer research, 74(13), 3603-3616.

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Multiparametric Flow Cytometry for Endothelial Cell Characterization

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For flow cytometry, the cells were stained for 15 min at 4 °C, in the dark in 100 μl MACS running buffer (Miltenyi Biotec) containing the antibodies detecting CD144 (BD Bioscience), CD31 (Biolegend), CD140b (BD Bioscience), CD309/KDR (Miltenyi Biotec), and CD45 (BD Bioscience). Afterwards, cells were washed with MACS running buffer and resuspended in 500 μl MACS running buffer. Flow cytometry was performed using a BD FACS Canto, and the data were analyzed with FlowJo software (Tree Star).

Thoma E.C., Heckel T., Keller D., Giroud N., Leonard B., Christensen K., Roth A., Bertinetti-Lapatki C., Graf M, & Patsch C. (2016). Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey. Scientific Reports, 6, 35830.

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ALDEFLUOR Assay for Cancer Stem Cells

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For the ALDEFLUOR assay (StemCell), dissociated single cells were suspended in assay buffer contain ALDEFLUOR substrate and incubated with or without DEAB. Analysis of tumor cell suspensions from xenograft tumors were performed as previous report. Briefly, PE-conjugated anti-mouse lineage antibodies (CD45 (BD), CD31 (BD), CD140b (BD), CD235a (BD), and H2KD (Biolegend)) were used for gating out non-breast cancer cells. For cell cycle analysis, cells were fixed with 70% alcohol at 4 °C overnight and stained with propidium iodide (Sigma-Aldrich) in the presence of 1% RNAase A (Takara) at 37 °C for 30 min prior to analysis. For apoptosis analysis, cells were stained with propidium iodide (Sigma-Aldrich) and Annexin V (BD), according to the manufacturer’s instructions. DCFH-DA (Sigma-Aldrich) was added to the cells without FBS for detecting ROS production. CytoFLEX (Beckman Coulter) was used for detection and data acquisition and analysis were performed in CytoExpert software. A MoFlo Astrios instrument (Beckman Coulter) was used for sorting cells.

Deng L., Gao X., Liu B., He X., Xu J., Qiang J., Wu Q, & Liu S. (2018). NMT1 inhibition modulates breast cancer progression through stress-triggered JNK pathway. Cell Death & Disease, 9(12), 1143.

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Pericyte Marker Characterization of MSCs

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Pericyte surface markers of ntMSCs and nbMSCs isolated from n = 3 patients were characterized by flow cytometry using fluorochrome‐conjugated anti‐human CD140a, CD140b, CD146, and CD90 (BD Biosciences, San Jose, California). The antibodies were incubated with MSCs for 60 minutes at room temperature, followed by three washes. MSCs were then analyzed using a MoFlo Astrios flow cytometer (Beckman Coulter, Inc., Pasadena, California) using the appropriate isotype‐matched and unstained controls.
We also measured the transcript expression of TBX18, recently determined to be expressed in the perivascular mural cells of mice vasculature,⁸ in human ntMSCs, nbMSCs, and abMSCs using qPCR (see below).

Wang S., Huang S., Johnson S., Rosin V., Lee J., Colomb E., Witt R., Jaworski A., Weiss S.J, & Si M. (2020). Tissue‐specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3‐ROBO1. Stem Cells Translational Medicine, 9(9), 1102-1113.

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Immunophenotyping of Mesenchymal Stem Cells

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For each marker, 10,000 MSCs were stained as described previously [20 (link)]. Cells were detached using TrypLE Express (Gibco) recombinant trypsin. TrypLE was deactivated using phosphate-buffered saline+2% FCS. Cells were incubated for 30 min at 4°C in the dark with human FcR blocking reagent (Miltenyi Biotec, Leiden, NLD) and the following antibodies: CD45-PE (#560975 BD Pharmigen, Breda, NLD), CD14 (#R0864, Dako, Heverlee, BEL), CD19 (130-091-328, Miltenyi), CD34 (BD #555821), CD73 (BD #550257), CD90 (#B113673 Biolegend, Fell, DE), CD105-Fitc (FAB 10971F, R&D, Minneapolis, MN, USA), and CD140b (BD #558821). Subsequently, cells were washed with PBS+2% FCS. Cell fluorescence was measured on a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). SYTOX Blue (Molecular Probes/Invitrogen, Eugene, OR, USA) was used for exclusion of dead cells.

van Rhijn-Brouwer F.C., van Balkom B.W., Papazova D.A., Hazenbrink D.H., Meijer A.J., Brete I., Briceno V., van Zuilen A.D., Toorop R.J., Fledderus J.O., Gremmels H, & Verhaar M.C. (2019). Paracrine Proangiogenic Function of Human Bone Marrow-Derived Mesenchymal Stem Cells Is Not Affected by Chronic Kidney Disease. Stem Cells International, 2019, 1232810.

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Multiparametric Flow Cytometry of PCs and PMVs

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PCs and PMVs were subjected to high sensitivity imaging flow-cytometry Amnis Image Stream MK II (ISX) (Amnis, America) and further analyzed by INSPIRE (v1.4.0). In brief, samples were stained with CD146 (1:100, BD, America), α-SMA (1:80, BD, America), CD140b (1:50, BD, America), NG (1:100, BD, America) and Annexin V (1:50, BD, America) for 30 min in the dark (RT). Annexin V binding buffer (BD, America) was used along with Annexin V antibody. Standard beads were added into MV samples (0.2 μm, 0.5 μm, 0.8 μm, Bangs laboratories, America) for the gate set and quantification of MVs.

Zhou H., Zheng D., Wang H., Wu Y., Peng X., Li Q., Li T, & Liu L. (2021). The protective effects of pericyte-derived microvesicles on vascular endothelial functions via CTGF delivery in sepsis. Cell Communication and Signaling : CCS, 19, 115.

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