Dynabead

Manufactured by Thermo Fisher Scientific

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Dynabeads are magnetic beads used in various laboratory applications. They are designed to efficiently capture and isolate target molecules, such as proteins, nucleic acids, or cells, from complex samples. The magnetic properties of Dynabeads allow for easy separation and manipulation of the captured targets.

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CD3/CD28 Dynabeads (277 citations) Dynabead mRNA Purification Kit (11 citations) Dynabead M-280 Streptavidin (7 citations) Dynabead Protein G magnetic beads (6 citations) Dynabead MyOne Streptavidin T1 (2 citations) MyOne™ Dynabead® Streptavidin C1 beads (2 citations) Dynabead-positive selection (2 citations) Dynabead MyOne Streptavidin T1 conjugated magnetic beads (2 citations) Dynabead® Mouse DC Enrichment kit (2 citations) MyOne™ Dynabead® Streptavidin C1 (1 citations)

2 112 protocols using dynabead

Isolation and Characterization of Murine Lung Microvascular Endothelial Cells

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Murine lung microvascular endothelial cells (MLMVEC) were isolated and cultured from murine lungs. Lungs were mechanically minced and enzymatically digested with 20 mg ml⁻¹ collagenase A (Roche, Basel, Switzerland) at 37 °C for 2 h. Dynabeads (Life Technologies, Carlsbad, CA, USA) were incubated with anti-PECAM-1 antibody (clone Mec13.3, 30 μg per 200 μl, purified from hybridoma supernatant) at 4 °C for 24 h. Lung homogenisates were incubated with Dynabeads at 4 °C for 45 min, followed by magnetic separation of the Dynabeads from the supernatant. MLMVEC cells were culture on gelatin-coated dishes for 4–7 days. The mRNA from MLMVEC was extracted using Trizol extraction (peqGOLD TriFast, Peqlab, Erlangen, Germany). mRNA was transcribed into cDNA using M-MLV reverse transcriptase. Quantitative expression analysis by qRT-PCR was performed using primers specific for murine ICAM-1 and GAPDH70 (link). Surface expression of ICAM-1 was analysed using immunofluorescence staining and flow cytometry as described previously9 (link).

Rossaint J., Kühne K., Skupski J., Van Aken H., Looney M.R., Hidalgo A, & Zarbock A. (2016). Directed transport of neutrophil-derived extracellular vesicles enables platelet-mediated innate immune response. Nature Communications, 7, 13464.

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Antibody-Mediated Protein Extraction

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This assay was performed as described previously (36 (link)). Briefly, antibody and Dynabeads (Invitrogen) were mixed in a reaction for 16 to 24 h. The extracted proteins were mixed with the Dynabeads carrying antibody and incubated for 1 h. The protein was separated from the Dynabeads via centrifugation and evaluated by Western blot analysis.

Xia X., Liu X., Chai R., Xu Q., Luo Z., Gu J., Jin Y., Hu T., Yu C., Du B., Huang H., Ou W., Liu S, & Liu N. (2021). USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells. The Journal of Biological Chemistry, 297(5), 101258.

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Purification and Analysis of CD71+ Erythroblasts

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CD71⁺ erythroblasts were purified from bone marrow by CD71 monoclonal antibody (Santa Cruz, sc-59112) conjugated to Dynabeads (Invitrogen, 11033) following manufacturers’ instructions. Anti-CD71 was incubated with Dynabeads on ice for 30–60 min and washed twice with Hank’s Balanced Salt Solution (HBSS) buffer (Gibco, 14025076). Bone marrow cells were incubated with Dynabead-conjugated antibodies on ice for 60 min, purified by magnetic separation and cells were washed twice with HBSS buffer. mRNA was isolated, converted to cDNA and Slc29a1 mRNA levels were quantified with the following primers: mouse 18S, forward 5′-GTAACCCGTTGAACCCCATT-3′ and reverse 5′-CCATCCAATCGGTAGTAGCG-3′; mouse Slc29a1, 5′-CTTGGGATTCAGGGTCAGAA-3′ and reverse 5′-ATCAGGTCACACGACACCAA-3′.

Zwifelhofer N.M., Cai X., Liao R., Mao B., Conn D.J., Mehta C., Keles S., Xia Y, & Bresnick E.H. (2020). GATA factor-regulated solute carrier ensemble reveals a nucleoside transporter-dependent differentiation mechanism. PLoS Genetics, 16(12), e1009286.

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PBMC Isolation and Stimulation for Autoimmune Therapy

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Human PBMCs were purified from blood using Lymphoprep (STEMCELL Technologies, Vancouver, BC, Canada) in SepMate PBMC isolation tubes (STEMCELL Technologies). Samples were collected at baseline, 5 months, and 12 months after beginning treatment. Purified PBMCs were frozen in liquid nitrogen with FBS (Atlas Biologicals, Ft. Collins, CO, USA) with 10% DMSO (Sigma, St. Louis, MO, USA) until all time point samples from each patient were collected. PBMCs were then thawed and stimulated (10⁵) with either Dynabeads (Gibco, Carlsbane, CA, USA) or rhGAD65 (5 μg/mL, Diamyd, Stockholm, Switzerland) in the presence of IL-2 (30 U/mL) in DMEM media (Life Technologies, Carlsbane, CA, USA) containing FBS (Atlas), human AB serum (Fisher, Hampton, NH, USA), and penicillin/streptomycin (Gibco) for 3 days (Dynabeads) or 7 days (rhGAD65). Cell culture supernatants were collected and cells were stored in RNAlater (Thermo Fisher, Waltham, MA, USA).

Heath K.E., Feduska J.M., Taylor J.P., Houp J.A., Botta D., Lund F.E., Mick G.J., McGwin G J.r., McCormick K.L, & Tse H.M. (2023). GABA and Combined GABA with GAD65-Alum Treatment Alters Th1 Cytokine Responses of PBMCs from Children with Recent-Onset Type 1 Diabetes. Biomedicines, 11(7), 1948.

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Isolation and Activation of Primary Human CD4 T Cells

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For the isolation of primary human CD4 T cells, human Buffy Coats from anonymous healthy donors were obtained from the Heidelberg University Hospital Blood Bank. CD4+ T cells were isolated by negative selection with the RosetteSepTM Human CD4+ T Cell Enrichment Cocktail and separated by Ficoll gradient centrifugation, resulting in homogenous populations of CD4+ T cells with a purity of 90–95% as assured by flow cytometry. Cells labeled as ‘Resting’ were cultured for 72 hr in complete RPMI media containing recombinant human IL2 (Biomol #155400.10) at 10 ng/ml final concentration. Whereas the cells labeled as ‘Activated’ were cultured for 72 hr in complete RPMI media containing recombinant human IL2 (Biomol #155400.10) at 10 ng/ml final concentration along with Dynabeads at a ratio of 25 µl human anti-CD3/28-labeled Dynabeads/10 million cells (#11132D, Gibco).

Sadhu L., Tsopoulidis N., Hasanuzzaman M., Laketa V., Way M, & Fackler O.T. (2023). ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells. eLife, 12, e82450.

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Activation and Expansion of T Cells

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CD4⁺ and CD8⁺ T cells from 4 independent donors were thawed, mixed at a 1:1 ratio, and rested for 3–4 hours in basal media (RPMI supplemented with 10% FBS) before activation. 3 × 10⁵ cells were seeded in a U-bottom 96-well plate at a density of 1.5 × 10⁶ cells/mL. Supported lipid bilayer formulations (aAPCs) and CD3/CD28 Human T Activator Beads (DynaBeads, Gibco) were suspended in complete T cell medium and cocultured with T cells at defined aAPC-to-cell ratios based on equivalent particle surface area of 65 ± 15 μm²/cell (1:3 microsphere-to-cell, 1:10 HeLa-to-cell, 1:1 RBC-to-cell, and 1:1 DynaBead-to-cell). For outgrowth experiments, media was exchanged for fresh cytokine-supplemented medium every 3 days following activation. On days 6, 9, 12, and 14 post-activation, all aAPC-stimulated cells were counted by hemocytometer. Cells were passaged into larger culture vessels once reaching densities > 1.25 × 10⁶/mL.

Olden B.R., Perez C.R., Wilson A.L., Cardle I.I., Lin Y.S., Kaehr B., Gustafson J.A., Jensen M.C, & Pun S.H. (2018). Cell-templated Supported Lipid Bilayers for T Cell Activation. Advanced healthcare materials, 8(2), e1801188.

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Jurkat and Primary T Cell Labeling and Transduction

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The Jurkat T cell line was obtained commercially (#TIB-152, ATCC, Manassas, VA) for initial nanoemulsion cell labeling characterizations. Jurkat cells were grown in RPMI-1640 media (Gibco, Waltham, MA) plus 10% fetal bovine serum (FBS), 10 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1 mM sodium pyruvate and 1.5 mg/mL sodium bicarbonate.
Primary human T cells were obtained from blood samples sourced from the San Diego Blood Bank and enriched for T cells by Ficoll (Histopaque 1077, Sigma Aldrich) gradient density centrifugation and magnetic assisted cell sorting (Dynabeads, Thermo Fisher). T-cells were then activated with human T-activator CD3/CD28 Dynabeads and allowed to expand for two days in RPMI-1640 supplemented with 10% FBS and 100 units/mL of recombinant human interleukin 2 (IL-2, Peprotech, Rocky Hill, NJ). For transduction, we employed a vector specific to epidermal growth factor receptor variant III (EGFR-vIII) as described by Johnson et al. (30 ). A detailed CAR virus production and human T cell transduction is available elsewhere (31 (link)). CAR receptor expression was confirmed by flow cytometry. We used T cell populations with >70% CAR+ expression.

Hingorani D.V., Chapelin F., Stares E., Adams S.R., Okada H, & Ahrens E.T. (2019). Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine-19 MRI detection. Magnetic resonance in medicine, 83(3), 974-987.

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In vitro Culture of P. falciparum NF54 Parasites

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P. falciparum NF54 parasites (27 (link)) were grown in vitro in O Rh⁺ erythrocytes at 37°C in a controlled atmosphere, using complete culture medium (RPMI 1640 supplemented with 0.5% AlbuMax II [Life Technologies BV, Nærum, Denmark]), essentially as described previously (28 (link)). The P. falciparum NF54-derived and pVBH-transfected clone G6 was generated as described elsewhere (29 (link)– (link)31 (link)). It was maintained in the same way as the parental strain P. falciparum NF54, except that blasticidin (10 mg/ml; Life Technologies) was added to shut down transcription of endogenous var genes and to erase the epigenetic memory. IEs were selected for surface expression of defined PfEMP1 proteins by immunomagnetic selection using (i) PfEMP1-specific rat antisera followed by biotinylated anti-rat antibody (Dako) and streptavidin-coupled Dynabeads (Fisher Scientific) or (ii) PAM1.4 followed by protein A-coupled Dynabeads (Fisher Scientific), essentially as described previously (32 (link)). For selection of IgM-binding IEs, we used human IgM (Sigma) coupled to M-450 epoxy beads (Life Technologies) according to the manufacturers' instructions. The genotypic identity of the parasites and the absence of Mycoplasma contamination were verified regularly as described previously (33 (link)).

Jeppesen A., Ditlev S.B., Soroka V., Stevenson L., Turner L., Dzikowski R., Hviid L, & Barfod L. (2015). Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fcμ. Infection and Immunity, 83(10), 3972-3981.

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Binding Assay of mTLR9 with DNA Oligonucleotides

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For the binding studies, a soluble mTLR9 ectodomain fused to the Fc fragment of mouse IgG (mTLR9-Fc, R&D Systems) was bound to protein-G-coupled Dynabeads (Dynabeads Protein G for Immunoprecipitation, Novex; cat. no. 10003D). Briefly, 7.5 μg of mTLR9-Fc (or PBS alone—negative control) was incubated with 70 μl of beads for 3 h at room temperature and washed three times with wash buffer (1 × PBS, 0.02% Tween-20, pH 7.5 or pH 6.0). Then, minM80^PD-3′FITC (250 pmol) and/or TCGTT^PD-3′FITC or TTTTT^PD-3′FITC (5 μmol) in phosphate buffer (pH 6 or 7.5) were added per sample, incubated for 3 h at room temperature, and washed three times with wash buffer of an appropriate pH. Bound DNA and mTLR9-Fc were eluted via incubation at 95 °C for 10 min in 42 μl formamide loading buffer. The ODNs and standard were separated using 20% TBE-urea polyacrylamide gel in TBE buffer and imaged using an IVIS Lumina (PerkinElmer). Then, mTLR9-Fc was detected with western blot analysis using goat anti-mouse-HRP antibodies (1:4,000, Santa Cruz Biotechnologies, sc-2005).

Pohar J., Lainšček D., Ivičak-Kocjan K., Cajnko M.M., Jerala R, & Benčina M. (2017). Short single-stranded DNA degradation products augment the activation of Toll-like receptor 9. Nature Communications, 8, 15363.

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Immunoprecipitation of GFP-ClC-2 in MDCK Cells

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MDCK cells expressing GFP-ClC-2 were lysed at 4°C with lysis buffer (1% Triton X-100, 25 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol) and a cocktail of protease inhibitors. The lysate was precleared by centrifugation at 10,000 rpm for 15 min and the supernatant incubated with 1.5 μg of mouse anti–γ-adaptin antibody absorbed to 50 μl of Dynabeads (Life Technologies, Carlsbad, CA) for 3 h at 4ºC. The immunocomplexes were washed five times with lysis buffer and once with wash buffer in Dynabead kit and eluted with 3.6× Laemmli sample buffer. The samples were heated at 70°C for 10 min, resolved by 4-12% SDS–PAGE, and processed for Western blot with either primary mouse anti–γ-adaptin or chicken anti-GFP antibodies, followed by secondary antibodies labeled with red IRDye⁶⁸⁰ (anti-mouse) and green IRDye⁸⁰⁰ (anti-chicken) and imaged using Odyssey.

de la Fuente-Ortega E., Gravotta D., Bay A.P., Benedicto I., Carvajal-Gonzalez J.M., Lehmann G.L., Lagos C.F, & Rodríguez-Boulan E. (2015). Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1. Molecular Biology of the Cell, 26(9), 1728-1742.

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