Dynabeads

Manufactured by BD

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Dynabeads are magnetic beads used for a variety of laboratory applications. They are designed for efficient separation, isolation, and purification of various biological molecules such as proteins, nucleic acids, and cells.

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17 protocols using dynabeads

Isolation and Culture of Murine Vascular Smooth Muscle Cells and Bone Marrow-Derived Macrophages

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Murine VSMCs were isolated from the aorta by enzyme isolation as previously described (27 (link)). Cultures were trypsinized, followed by the addition of CD31-conjugated Dynabeads (Pharmingen) to the cell suspension and magnetic separation of endothelial cells (ECs) from VSMCs. Selectivity of recovered VSMC populations was highly specific based on detection of α-smooth muscle actin protein by FACS analysis in the VSMC cultures (Supplementary Figure 1). VSMCs at passage 2 to 10 were used for experiments. Murine VSMCs were grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/ml; Invitrogen Life Technologies, Carlsbad, CA), and streptomycin sulfate (Invitrogen Life Technologies). Mouse bone marrow cells were prepared from the femur and tibia of 6-week old female mice and cultured with 30 ng/ml macrophage colony stimulation factor (M-CSF) (Peprotech, Rocky Hill, NJ, USA) for 3 days to prepare bone marrow-derived macrophages (BMMs) as previously described (28 (link)). BMMs were grown in Minimal Essential Media (Thermo Fisher Scientific) supplemented with 10% FBS and 30 ng/ml M-CSF.

Choi B., Shin M.K., Kim E.Y., Park J.E., Lee H., Kim S.W., Song J.K, & Chang E.J. (2019). Elevated Neuropeptide Y in Endothelial Dysfunction Promotes Macrophage Infiltration and Smooth Muscle Foam Cell Formation. Frontiers in Immunology, 10, 1701.

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Isolation of Mouse Lung Endothelial Cells

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Mouse lung ECs were isolated as previously described^{23 (link)}. Klf2^+/− mice (B6; 129S4-Klf2^tm1.1Hhn/J, Stock# 026926) were obtained from The Jackson Laboratory. Briefly, lungs from three Klf2^+/− or three Klf2^+/+ mice were minced into pieces. The lung pieces were digested using pre-warmed (37 °C) type I collagenase (#9001-12-1, Gibco) in a cell incubator at 37 °C for 45 min. The cell suspension was then filtered and centrifuged at 1200 rpm. The precipitates were resuspended in PBS containing BSA and penicillin/streptomycin and added CD31 (BD Pharmingen™, #553370) coated Dynabeads™ (Invitrogen) to incubate on a rotor at room temperature for 15 min. The cells in EP tubes were put on Magnetic Separation Rack and washed with growth medium (DMEM + 20%FBS + penicillin/streptomycin + heparin) for 4 times. Cells were resuspended in growth medium and grown in 0.1% gelatin coated 6-well plate. When the cells approached confluence, CD102 (BD Pharmingen™, #553326) coated Dynabeads™ sorting were performed. Then, the isolated cells were used to do the experiments. All animal procedures conformed to the Guideline for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center.

Xu Y., Liu P., Xu S., Koroleva M., Zhang S., Si S, & Jin Z.G. (2017). Tannic acid as a plant-derived polyphenol exerts vasoprotection via enhancing KLF2 expression in endothelial cells. Scientific Reports, 7, 6686.

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Isolation and Characterization of Mouse Lung Endothelial Cells

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KLF2^+/− mice (B6; 129S4‐Klf2^tm1.1Hhn/J, Stock# 026926) were purchased from JAX Laboratory (Bar Harbor, ME). Mouse lung ECs were isolated as described previously.26 Briefly, lungs from 3 mice of each group were minced into pieces and digested with prewarmed type I collagenase (2 mg/mL) at 37°C for 45 minutes. The suspension was filtered through a 70‐μm disposable cell strainer and then centrifuged at 800 g at 4°C for 5 minutes. Precipitates were resuspended in cold PBS+BSA+Penicillin/Streptomycin. The cell suspension was then incubated with CD31 (#553370; BD Pharmingen, San Jose, CA)‐coated Dynabeads (Invitrogen, Carlsbad, CA) on a rotor at room temperature for 15 minutes, and then washed until the suspension became visibly clear. Cells with beads were resuspended in growth medium (DMEM+20%FBS+penicillin/streptomycin+heparin) and plated on 0.1% gelatin‐coated culture dishes. At 5 to 9 days after plating, cells approached confluence. A second sort of cells were performed with CD102‐coated (#553326; BD Pharmingen) Dynabeads sorting. Then, cells were cultured in growth medium and used for indicated experiments immediately.

Xu Y., Xu S., Liu P., Koroleva M., Zhang S., Si S, & Jin Z.G. (2017). Suberanilohydroxamic Acid as a Pharmacological Kruppel‐Like Factor 2 Activator That Represses Vascular Inflammation and Atherosclerosis. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(12), e007134.

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Ex Vivo CD8+ T Cell Proliferation

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For preparing PBMCs, 10 ml of whole blood was drawn from each donor and separated by Ficoll gradient centrifugation. The PBMC layer was collected and washed twice with PBS. The isolation of CD8⁺ T cells from PBMCs was performed by negative selection using EasySep Human T-Cell Isolation Kit (StemCell Technologies, Canada). The examination of the ex vivo proliferation of isolated CD8⁺ T cells was based on labeling cells with eFluor670 proliferation tracer (eBioscience). The labelled cells were then stimulated with Dynabeads (Invitrogen) coated with anti-CD3 (BD Biosciences) in the presence and absence of different TLR agonists, or Dynabeads coated with anti-CD3 plus anti-CD28 (BD Biosciences) as a positive control. On day 7, the cells were analyzed by flow cytometry (BD LSRFortessa) and the percentage of proliferated cells was determined according to the eFluor670 dilution. To determine the dosage effect of TLR agonists, co-stimulation was carried out under three concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml.

Qiu C., Wang J., Zhu L., Cheng X., Xia B., Jin Y., Qin R., Zhang L., Hu H., Yan J., Zhao C., Zhang X, & Xu J. (2022). Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors. Frontiers in Bioengineering and Biotechnology, 10, 1027619.

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Endothelial Cell Culture Conditions

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BAECs were grown in DMEM containing 10% FBS and penicillin/streptomycin. HUVECs were grown in DMEM/F12, 10% FBS, 5 mg ml⁻¹ ECGS, 100 μg ml⁻¹ heparin, penicillin/streptomycin or EGM media (Lonza). Mouse endothelial cells were grown in DMEM containing 20% FBS, 1× non-essential amino acids (Gibco), 2 mM L-glutamate, 50 μg ml⁻¹ gentamicin, 4 μg ml⁻¹ amphotericin B, 100 μg ml⁻¹ heparin, 5 mg ml⁻¹ ECGS and penicillin/streptomycin. Primary mouse endothelial cells were isolated from the lung, using rat anti-mouse CD31 antibody (clone MEC13.3, Pharmingen, no. 553370) and Dynabeads (cat. no. 110.35, Invitrogen) as previously described^{64 (link)}. No cell lines were used in this study.

Yun S., Budatha M., Dahlman J.E., Coon B.G., Cameron R.T., Langer R., Anderson D.G., Baillie G, & Schwartz M.A. (2016). Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling. Nature cell biology, 18(10), 1043-1053.

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Isolation and Characterization of Primary Mouse Lung Endothelial Cells

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The primary mouse lung endothelial cells (MLEC) were isolated from a 10-week-old, female, Balb/c mice and were cultured as described elsewhere^{29 (link)}. Briefly, freshly isolated mouse lung was minced using autoclaved scissors, digested by collagenase I, and filtered through a 70-μm cell strainer. The cell suspension was incubated with anti-rat Dynabeads (Thermo Fisher Scientific, MA, USA, 11035) conjugated with anti-mouse CD31 antibody (BD Biosciences, NJ, USA, 557355). Pooled cells were seeded in a 12-well-plate pre-coated with 0.1% gelatin. Upon reaching confluence, the cells were trypsinized and then incubated with anti-mouse ICAM-2 antibody (BD Biosciences, NJ, USA, 553326) conjugated Dynabeads. Pooled cells were seeded in 12-well-plates pre-coated with 0.1% gelatin. Harvested cells were assessed using tube formation assay, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate Low Density Lipoprotein (DiI-Ac-LDL) uptake, and CD31 gene expression (Fig. S4).

Gulay K.C., Aoshima K., Maekawa N., Suzuki T., Konnai S., Kobayashi A, & Kimura T. (2022). Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages. Scientific Reports, 12, 2124.

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Exosome Isolation and Characterization

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Exosomes were isolated by the differential centrifugation method.²² Briefly, the GSC culture medium was centrifuged at 300 g for 10 min to pellet cells, filtered through 0.22 µm bottle-top vacuum system (Corning), then centrifuged at 2000 g for 10 min to remove dead cells. Cell debris was removed by centrifugation at 10,000 g for 30 min. Exosomes were pelleted by ultracentrifuge at 100,000 g for 70 min and washed with PBS once, then pelleted again by ultracentrifuge at 100,000 g for another 70 min. The exosome pellet was resuspended in PBS for subsequent tests. Exosome numbers were quantified using a NanoSight NS300. 2 × 10¹⁰ exosomes were added to 1 × 10⁶ immune cells. Pre-enriched exosomes were resuspended in PBS and bound to CD63-coated Dynabeads (Thermo Fisher Scientific) during an overnight incubation. The following day the Dynabeads-bound exosomes were stained with IgG control antibodies, CD63-FITC, and CD9-Brilliant Violet 510 (BD Biosciences) and analyzed by a Gallios 561 flow cytometer (Beckam Coulter, Indianapolis, IN).

Gabrusiewicz K., Li X., Wei J., Hashimoto Y., Marisetty A.L., Ott M., Wang F., Hawke D., Yu J., Healy L.M., Hossain A., Akers J.C., Maiti S.N., Yamashita S., Shimizu Y., Dunner K., Zal M.A., Burks J.K., Gumin J., Nwajei F., Rezavanian A., Zhou S., Rao G., Sawaya R., Fuller G.N., Huse J.T., Antel J.P., Li S., Cooper L., Sulman E.P., Chen C., Geula C., Kalluri R., Zal T, & Heimberger A.B. (2018). Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes. Oncoimmunology, 7(4), e1412909.

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Isolation of Endothelial Cells from Dental Pulp

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Dental pulps were incubated for 30 min at 37°C in DMEM containing 1% collagenase D (from Clostridium histolyticum; Sigma-Aldrich), 1 U/ml dispase (Thermo Fisher Scientific), and 1 U/ml DNase (Invitrogen) before cells were dissociated by gentle trituration. Cells were isolated using Dynabeads (Veritas), according to the manufacturer’s instructions. To isolate ECs, cells were incubated with an anti-CD31 antibody (BD Biosciences) preconjugated to Dynabeads (M-450) anti-Rat IgG, and positively selected as CD31⁺ cells.

Matsubara T., Iga T., Sugiura Y., Kusumoto D., Sanosaka T., Tai-Nagara I., Takeda N., Fong G.H., Ito K., Ema M., Okano H., Kohyama J., Suematsu M, & Kubota Y. (2022). Coupling of angiogenesis and odontogenesis orchestrates tooth mineralization in mice. The Journal of Experimental Medicine, 219(4), e20211789.

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Immortalized Mouse Lung Endothelial Cells

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Lung tissues from three 7–10 day old mice were removed aseptically and rinsed in ice-cold DMEM. After removal of the larger blood vessels, the lungs were minced into ~1×2-mm squares and digested with 1.5mg/ml collagenase A (Roche), at 37°C for 45 minutes on a rotator. The cellular digest was filtered through sterile 70μm nylon mesh and the mesh washed with 15ml 10% FBS-DMEM media to stop digestion. Cells were centrifuged at 400xg for 10 minutes, resuspended in 0.1% BSA/PBS, incubated with anti-CD31-antibody (BD) coated Dynabeads (Invitrogrn), and then separated in a magnetic field. After washing away of unbound cells, remaining cells were seeded on 2% gelatin coated plates cultured with Endothelial cell medium (ScienCell). After 3–5 days, cells were separated again by anti-ICAM2 antibody (BD) coated Dynabeads. The cells were plated at a concentration of 300,000 cells/ml into gelatin-coated T-25 flasks, and subsequently split 1:2 at each passage. After 3 passages, cells were immortalized using lentivirus expressing SV40 large T antigen (Genecopoeia).

Shih Y.P., Sun P., Wang A, & Lo S.H. (2015). Tensin1 positively regulates RhoA activity through its interaction with DLC1. Biochimica et biophysica acta, 1853(12), 3258-3265.

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Isolating Mouse Microglia Using Anti-CD11b Microbeads

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To prepare the anti-CD11b microbeads, 25 μl of sheep anti-rat Dynabeads (Invitrogen™) were washed three times with 0.75 ml of RPMI 1640 medium using a magnetic scaffold, allowing 1 min for the beads to settle each time. The Dynabeads were then suspended in 0.5 ml of serum-free 1640 medium and incubated overnight at 4 °C on a rotator (continuous rotation at 8 RPM) with 8 μl of rat anti-mouse CD11b (BD Pharmingen™, cat. no. 553308). After incubation, the anti-CD11b-conjugated microbeads were washed twice, resuspended in 0.5 ml of 1640 medium, and mixed with 0.5 ml prepared mouse brain cells. The mixture was incubated on a rotator for 30 min at 4 °C and then placed on a magnetic scaffold for 1 min, after which the supernatant was removed. The resulting MG-bead mixtures from six mice were pooled as one sample and washed with PBS before being used for RNA purification.

Wang X., Liu L., Jiang X., Saredy J., Xi H., Cueto R., Sigler D., Khan M., Wu S., Ji Y., Snyder N.W., Hu W., Yang X, & Wang H. (2023). Identification of methylation-regulated genes modulating microglial phagocytosis in hyperhomocysteinemia-exacerbated Alzheimer’s disease. Alzheimer's Research & Therapy, 15, 164.

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