Cd146 microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

CD146 microbeads are a laboratory reagent used for the isolation and enrichment of CD146-positive cells from complex biological samples. CD146, also known as melanoma cell adhesion molecule (MCAM), is a cell surface marker expressed on various cell types, including endothelial cells, pericytes, and some stem cells. The CD146 microbeads allow for the specific capture and separation of CD146-positive cells, which can be useful for various research and diagnostic applications.

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Lab products found in correlation

Feeder removal microbeads, by Miltenyi Biotec (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Mgso4, by Carl Roth (1 mentions) Sodium chloride (nacl), by Carl Roth (1 mentions) Potassium chloride (kcl), by Carl Roth (1 mentions) Nah2po4, by Merck Group (1 mentions) Liberase th, by Roche (1 mentions) Pronase, by Merck Group (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Nycodenz, by Axis-Shield (1 mentions) Nk cell isolation kit 2, by Miltenyi Biotec (1 mentions) Liberase, by Roche (1 mentions) Anti mouse igg1 microbeads, by Miltenyi Biotec (1 mentions) Liver dissociation kit, by Miltenyi Biotec (1 mentions) Qiashredder, by Qiagen (1 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions) Rlt plus buffer, by Qiagen (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Facsaria, by BD (1 mentions)

15 protocols using cd146 microbeads

Isolation and Culture of Cardiac Cell Types

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For culture, cardiac fibroblasts were isolated from 6 to 8 week-old Gata4^flox/flox, Gata6^flox/flox, and Gata4^flox/floxGata6^flox/flox mice as previously described [34 (link)]. Whole hearts without atria were quickly cut into small pieces in ice-cold PBS. Digestion was performed using Liberase TH (Roche) with SADO buffer mix solution (20 mM HEPES–NaOH (Roth, Nr. 9105, pH 7.6), 130 mM NaCl (Roth, Nr. 9265), 3 mM KCl (Roth, Nr. 6781), 1 mM NaH2PO4 (Sigma, Nr. S5011), 4 mM Glucose (Roth, Nr. HN06), 3 mM MgSO4 (Roth, Nr. 2278) in sterile, filtered dH20). The digested cell solution was plated and after 3 h washed once with PBS and changed to DMEM supplemented with 10% FCS. Isolated fibroblasts were infected with AdCre or Adβ-Gal for 3 h and after two washing steps with PBS cultured in DMEM supplemented with 2% FCS for 48 h. Cells were passaged two times at maximum before harvesting.
For immediate RNA or protein-isolation, cardiac fibroblasts and cardiac endothelial cells were isolated from hearts of adult mice using CD146 microbeads and feeder removal microbeads with MACS (magnetic cell separation) technology from Miltenyi Biotec.
Adult ventricular cardiomyocytes were isolated as previously described using a Langendorff system [24 (link)].

Dittrich G.M., Froese N., Wang X., Kroeger H., Wang H., Szaroszyk M., Malek-Mohammadi M., Cordero J., Keles M., Korf-Klingebiel M., Wollert K.C., Geffers R., Mayr M., Conway S.J., Dobreva G., Bauersachs J, & Heineke J. (2021). Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis. Basic Research in Cardiology, 116(1), 26.

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Isolation of Primary Liver Cells

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Liver tissues were preserved in RPMI 1640 medium (Gibco, Karlsruhe, Germany) on ice immediately after surgical removal, and cells were isolated within 6 h. Nycodenz (Axis Shield, Rodelokka, Norway) gradients density centrifugation was performed to isolate primary liver KCs and hepatic stellate cells (HSCs). In brief, liver samples were cut into 1-5 mm thick slices and rinsed by phosphate-buffered saline (PBS). The tissue slices were digested with 15 mg/mL pronase (Sigma, St. Louis, USA) at 37 °C for 20 min. After two times of 80 μm and 60 μm nylon mesh filtration, the tissue solution was mixed with Nycodenz solution (16.7% for KCs and 28.7% for HSCs) and centrifuged (1400× g, 20 min) without the brake, dividing into three layers. We collected the middle layer and interfaces. To isolate sinusoidal endothelial cells (SECs), CD146 MicroBeads (Miltenyi Biotec, Teterow, Germany) and a magnetically activated cell sorting system were employed to filtrate the tissue digestive solution instead of Nycodenz. Cells were seeded in multiple well plates and cultured at 37 °C in 5% CO₂ in RPMI 1640 with 10% fetal calf serum (FCS, PAN, Aidenbach, Germany) and 1% Penicillin-Streptomycin (Sigma, St. Louis, MO, USA). Detailed procedures were described previously [20 (link),21 (link),22 (link)].

Lin H., Fan Y., Wieser A., Zhang J., Regel I., Nieß H., Mayerle J., Gerbes A.L, & Steib C.J. (2021). Albumin Might Attenuate Bacteria-Induced Damage on Kupffer Cells for Patients with Chronic Liver Disease. Cells, 10(9), 2298.

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Isolation of Liver-Infiltrating Immune Cells

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Liver‐infiltrating lymphocytes (LILs) were isolated as described.⁽¹⁶⁾ Liver nonparenchymal cells were obtained by digestion in a medium containing 0.1% Liberase (Roche, Switzerland), as described.⁽¹⁷⁾ LSECs were isolated by positive isolation with CD146 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Intrahepatic NK cells were isolated by the NK Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of isolated LSECs and NK cells was >90% after isolation.

Du Y., Yan H., Zou S., Khera T., Li J., Han M., Yang X., Wang B., Liu J., Sun S., Zheng X., Dittmer U., Lu M., Yang D., Wedemeyer H, & Wu J. (2021). Natural Killer Cells Regulate the Maturation of Liver Sinusoidal Endothelial Cells Thereby Promoting Intrahepatic T‐Cell Responses in a Mouse Model. Hepatology Communications, 5(5), 865-881.

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Purification of CD146+ Bone Marrow Cells

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CD146⁺ cells were purified from BM by using CD146 MicroBeads (130-092-007, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. Cell sorting efficiency was further verified by flow cytometry.

Ma Y., Chang N., Liu Y., Liu F., Dong C., Hou L., Qi C., Yang L, & Li L. (2021). Silencing IQGAP1 alleviates hepatic fibrogenesis via blocking bone marrow mesenchymal stromal cell recruitment to fibrotic liver. Molecular Therapy. Nucleic Acids, 27, 471-483.

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Isolation and Expansion of Endometrial Stem Cells

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Endometrial stem cells were isolated by previously described magnetic beading method [9 (link)]. In short, stromal cells were first incubated with PE-conjugated anti-CD140b (R&D System, Minneapolis, MN, USA) antibody followed by anti-mouse IgG1 microbeads (Miltenyi Biotec Inc.). After incubation, cells were applied to Miltenyi MS columns with magnetic fields to isolate CD140b⁺ population. Afterwards, cells were expanded for 7 days followed by another round of beading with CD146 microbeads (Miltenyi Biotec Inc.). CD140b⁺CD146⁺ population (endometrial stem-like cells, ESCs) were subjected to following experiments.

Xue X., Li X., Yao J., Zhang X., Ren X, & Xu S. (2022). Transient and Prolonged Activation of Wnt Signaling Contribute Oppositely to the Pathogenesis of Asherman’s Syndrome. International Journal of Molecular Sciences, 23(15), 8808.

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Isolation and Purification of Murine LSECs

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Murine livers were obtained and dissociated using the liver dissociation kit (Miltenyi Biotec) in accordance with the manufacturer's instructions. Dissociated hepatic cells were passed through a 40-µm filter, and red blood cells (RBCs) were lysed by RBC lysis buffer (eBioscience). The cell suspension was washed with PEB buffer (0.5% [v/v] BSA, 5 mM EDTA in PBS) before incubating in CD146 MicroBeads (Miltenyi Biotec) for 15 min at 4°C. LSECs were enriched by passing the cell suspension through the MACS separator unit (Miltenyi Biotec). The enriched LSEC suspension was washed several times with PEB buffer and stained with Fc receptor blocker (antimouse FCGR3/CD16⁺-FCGR2B/CD32; Biolegend 101301, RRID:AB_312800). Subsequently, cells were stained with anti-CD31-PE (1:50; Thermo Fisher Scientific 12-0311-82, RRID:AB_465632) for 30 min at 4°C and then stained with DAPI (1:1000; Sigma D8417). Cells were sorted using a FACS Aria or Influx (BD Bioscience) directly into RLT plus buffer (Qiagen), passed through a QIAshredder (Qiagen), and stored at −80°C until use.

Yin K., Patten D., Gough S., de Barros Gonçalves S., Chan A., Olan I., Cassidy L., Poblocka M., Zhu H., Lun A., Schuijs M., Young A., Martinez-Jimenez C., Halim T.Y., Shetty S., Narita M, & Hoare M. (2022). Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance. Genes & Development, 36(9-10), 533-549.

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T Cell Isolation and Differentiation

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T cells were purified from PB cells prepared from fresh leukopaks as described above. Naive CD4⁺ T cells were negatively selected using a naive CD4⁺ T cell isolation kit (Miltenyi Biotec). Total CD4⁺ and CD8⁺ T cells were isolated using CD4⁺ or CD8⁺ T cell isolation kits (Miltenyi Biotec). Tcons were positively selected with CD4 microbeads after depletion of CD25 cells with CD25 microbeads (Miltenyi Biotec). CD4⁺CD146⁺ and CD4⁺CD146⁻ T cells were purified using CD146 microbeads (Miltenyi Biotec) from negatively selected total CD4⁺ T cells. Purified T cells were activated with anti-CD3/CD28 or anti-CD3/ICOS antibody–coated Dynabeads M-450 Tosylactivated (Invitrogen) at a bead/cell ratio of 1:5. For Th1 differentiation, IL-2 (2 ng/ml), IL-12 (10 ng/ml), and neutralizing antibodies against IL-4 (10 μg/ml) were added. All antibodies were obtained from eBioscience unless otherwise indicated. For Th17 differentiation, IL-1β (20 ng/ml), IL-6 (30 ng/ml), IL-23 (30 ng/ml), TGF-β (2 ng/ml), and neutralizing antibodies against IL-4 (5 μg/ml) and IFN-γ (2 μg/ml) were added. Exposure to differentiating cytokines (all from R&D Systems) and antibodies was maintained throughout the 7-day culture period.

Li W., Liu L., Gomez A., Zhang J., Ramadan A., Zhang Q., Choi S.W., Zhang P., Greenson J.K., Liu C., Jiang D., Virts E., Kelich S.L., Chu H.W., Flynn R., Blazar B.R., Hanenberg H., Hanash S, & Paczesny S. (2016). Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease. JCI Insight, 1(6), e86660.

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Enrichment and Transplantation of CD146+ Bone Marrow Cells

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CD146⁺ cells were enriched and depleted from EGFP⁺ and EGFP⁻ bone marrow, respectively, by using CD146 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. About 0.35% CD146⁺ cells were separated from EGFP⁺ bone marrow and 92.44% CD146⁻ cells were separated from EGFP⁻ bone marrow complementarily. Cell sorting efficiency was further verified by flow cytometry. Immunomagnetic cell-sorted EGFP⁺/CD146⁺ cells and EGFP⁻/CD146⁻ cells were mixed at the original ratio for transplantation.

Yang L., Dong C., Yang J., Yang L., Chang N., Qi C, & Li L. (2019). MicroRNA-26b-5p Inhibits Mouse Liver Fibrogenesis and Angiogenesis by Targeting PDGF Receptor-Beta. Molecular Therapy. Nucleic Acids, 16, 206-217.

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Isolation and Culture of Mouse Liver Sinusoidal Endothelial Cells

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Livers of WT mice were perfused with 0.05% GBSS/collagenase (from clostridium histolyticum; Sigma Aldrich) solution, removed, and digested in GBSS/collagenase at 37 °C for 20 min. After filtration through a steel mesh, liver cells were washed twice with GBSS. Non-parenchymal cells were removed by density gradient centrifugation, using a 30% Nycodenz solution (Progen Biotechnik, Heidelberg, Germany). By magnetic-activated cell sorting (MACS), CD146⁺ LSECs were isolated using CD146 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, LSECs were seeded onto collagen-coated 24-well plates in DMEM (high glucose) supplemented with 8% FCS (Sigma Aldrich), 2% L-glutamine (Life Technologies, Carlsbad, CA, USA), and 1% penicillin/streptomycin (ThermoFisher Scientific) for 2 days to form a monolayer.

Linke A., Cicek H., Müller A., Meyer-Schwesinger C., Melderis S., Wiech T., Wegscheid C., Ridder J., Steinmetz O.M., Diehl L., Tiegs G, & Neumann K. (2022). Antigen Cross-Presentation by Murine Proximal Tubular Epithelial Cells Induces Cytotoxic and Inflammatory CD8+ T Cells. Cells, 11(9), 1510.

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Isolation and Characterization of Intestinal and Endothelial Cells

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Small intestine mucosa was first isolated from Villin Cre^ER(+/-)EGFR and Villin Cre^ER(+/-)HIF1α mouse lines as previously described[35 (link)]. EGFR and HIF1α deficiency were then confirmed via RT-PCR using total RNA extracted from enterocytes. For endothelial specific EGFR deficiency confirmation in the Tie-2-CreER(+/-)EGFR(flox/flox) mouse line, lung endothelial cells were used to verify the efficiency of gene ablation as previously described[36 (link)]. In brief, lung tissue was harvested and enzymatically digested (2mg/mL Collagenase I (Gibco 17100–017)) in Live Cell Imaging Solution (Invitrogen, A14291DJ) in a GentleMACS dissociator. Homogenates were filtered to remove undigested tissue and resuspended. Endothelial cells were then isolated from this lung cell suspension using CD146 MicroBeads (#130-092-007) and a MS Column (Miltenyi Biotec, Auburn, CA). Subsequently, total RNA was extracted from isolated endothelial cells and EGFR deficiency was verified via RT-PCR as detailed below.

Onufer E.J., Aladegbami B., Imai T., Seiler K., Bajinting A., Courtney C., Sutton S., Bustos A., Yao J., Yeh C.H., Sescleifer A., Wang L.V., Guo J, & Warner B.W. (2020). EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis. PLoS ONE, 15(9), e0236964.

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