Magnetic bead

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, France, Japan, China

Magnetic beads are small, spherical particles that are coated with a magnetic material. They can be used for a variety of laboratory applications, such as cell separation, protein purification, and nucleic acid isolation.

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Lab products found in correlation

547 protocols using magnetic bead

IEL Survival Assay with Osteopontin

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CD45⁺ IEL from Spp-1^−/− mice were positively selected using magnetic beads (Miltenyi) and cultured (1×10⁵ cells/well) in a 96-well flat-bottomed well plate in RPMI complemented with 10% fetal bovine serum, penicillin/streptomycin, HEPES, L-glutamine and β-mercaptoethanol. These are “read out” cells. Then, these cells were cultured in the presence (1×10⁵ cells/well) or absence of magnetic bead-enriched (Miltenyi) iCD8α cells from Rag-2^−/− mice, or TCRβ⁺ or TCRγδ⁺ IEL from CD45.1 WT mice. For normalization purposes, the purity of these cells was determined by flow cytometry. Anti-osteopontin antibodies (2 μg/ml) (R&D; clone AF808) were added to some wells. After 4 h of incubation, cells were recovered and stained for surface markers, 7AAD and Annexin V. For all experiments, IEL were gated according to their size in a forward versus side scatter. Then, cells were selected by their expression of CD45.2 and gated for the individual subpopulations, followed by analysis of 7AAD incorporation and annexin V staining. Increased survival was determined as 100 - [% of annexin V⁺ read-out cells in co-culture x 100 / % of annexin V⁺ cells cultured alone]. Osteopontin concentration from these cultures was determined by an ELISA kit (R&D) following the manufacturer’s instructions.

Nazmi A., Greer M.J., Hoek K.L., Piazuelo M.B., Weitkamp J.H, & Olivares-Villagómez D. (2020). Osteopontin and iCD8α cells promote intestinal intraepithelial lymphocyte homeostasis. Journal of immunology (Baltimore, Md. : 1950), 204(7), 1968-1981.

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Differentiation of Osteoclasts from iPSC-Derived Hematopoietic Cells

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CD45⁺ cells were purified from iPSC-derived hematopoietic cells by magnetic beads (Miltenyi Biotec) and were cultured for a week in Minimal Essential Medium Alpha Modification (αMEM, Gibco), 10% FBS, 100 U/ml P/S, 2 mM L-glutamine in presence of 20 ng/ml m-SCF and 100 ng/ml m-M-CSF (PeproTech). Cells were subsequently transferred either on flat-bottomed 96-well tissue culture plates or on dentine slices (Osteosite, iDS, Pantec) and cultured in the same medium supplemented with 20 ng/ml m-M-CSF and 30 ng/ml m-RANKL (PeproTech). When osteoclasts were visible on tissue culture plates (after 5–10 days of culture), they were fixed and stained with tartrate resistance acid phosphatase (TRAP) assay (Sigma-Aldrich: acid phosphatase, leukocyte) accordingly to manufacturer’s instructions, while dentine slices were stained with 1% toluidine blue for 3 min and then washed in water. A similar protocol was pursued with oc/oc or WT mice freshly isolated splenocytes or BM cells, after purification of CD11b⁺ (instead of CD45⁺) cells by magnetic beads (Miltenyi Biotec).

Neri T., Muggeo S., Paulis M., Caldana M.E., Crisafulli L., Strina D., Focarelli M.L., Faggioli F., Recordati C., Scaramuzza S., Scanziani E., Mantero S., Buracchi C., Sobacchi C., Lombardo A., Naldini L., Vezzoni P., Villa A, & Ficara F. (2015). Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts. Stem Cell Reports, 5(4), 558-568.

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Isolation and Co-culture of CD11c+ DCs and CD4+ T cells

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CD11c⁺ DCs were isolated from the spleen of C. muridarum-immunized mice on day 7 p.i. or naïve C57BL/6 mice using magnetic beads and MACS columns through positive selection (Miltenyi Biotec, Auburn, CA, USA) as described above. CD4⁺ T cells were isolated from the spleen of C. muridarum-immunized mice on day 7 p.i. or naïve C57BL/6 mice using magnetic beads and MACS columns through negative selection (Miltenyi Biotec, Auburn, CA, USA) as described previously [24 (link)]. Briefly, spleen single-cell suspensions were mixed with Biotin-Antibody Cocktail, then incubated with Anti-Biotin MicroBeads. Labeled cells passed through magnetic columns for negative selection of CD3⁺ CD4⁺ T cells. As shown by flow cytometric analysis, the purity of the CD4⁺ T cells was more than 96%. CD4⁺ T cells were co-cultured with CD11c⁺ DCs (DC/T cell ratio, 1:5; CD11c⁺ DCs: 1 × 10⁵, CD4⁺ T cells: 5 × 10⁵) in 96-well plates in the presence or absence of UV-sterilized C. muridarum (UV-Cm) for 48h. Cell supernatants were collected for analyzing IFN-γ, IL-4, and IL-17 production by ELISA.

Zeng J., Yang S., Tuo Y., Zha X., Sun R., Lu T., Zhang H., Tan L., Qiao S, & Bai H. (2023). IL-27 Signaling Promotes Th1 Response by Downregulating IL-10 Production in DCs during Chlamydial Respiratory Infection. Microorganisms, 11(3), 604.

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Myeloid Cell-Mediated CD8+ T Cell Activation

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CD11b⁺ myeloid cells, that were positively selected using magnetic beads (Miltenyi) from young or adult tumors, were used ad APCs. The number of APCs was normalized to 100K mCherry⁺ cells per well, in a 96-well flat-bottom cell culture plate. CD8⁺ T cells from naïve P14 mice that were negatively selected using magnetic beads (Miltenyi) were used as responders at the indicated APC-to-P14 ratio. For proliferation assay shown in Fig. S6G, the purified CD8⁺ T cells were labeled with CTV at 5 μM final concentration. Cells were incubated for 1 or 3 days as described at 37°C in a CO₂ incubator.
After the incubation period cell aggregates were disrupted and detached from the plate by adding 2 mM EDTA for 10 min at RT. The single cell suspension was stained for the indicated cell surface or intracellular markers and analyzed by flow cytometry. As positive control, naïve CD8⁺ T cells were stimulated with 1 μM GP33 synthetic peptide.

Moustaki A., Crawford J.C., Alli S., Fan Y., Boi S., Zamora A.E., McDonald N.M., Wu G., Nakitandwe J., Newman S., Foy S., Silkov A., Thomas P.G., Pappo A., Dyer M.A., Stewart E., Federico S, & Youngblood B. (2022). Antigen cross-presentation in young tumor-bearing hosts promotes CD8+ T cell terminal differentiation. Science immunology, 7(68), eabf6136.

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Adoptive Transfer of OT-1 T Cells

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Naïve OT-1 cells were purified by negative selection using magnetic beads (Miltenyi Biotec). 10⁴ OT-1 (CD45.1) cells were adoptively transferred into naïve C57BL6/J (CD45.2) animals in 500 uL DMEM i.v. For adoptive transfer experiments, mice harboring memory OT-1 cells primed with viral vectors were sacrificed, splenocytes were isolated using a standard protocol, and CD8 T cells were purified using magnetic beads (Miltenyi Biotec). Following magnetic separation, OT-1 (CD45.1) cells were stained with CD45.1 (Invitrogen) antibody and sorted using FACSAria II (BD) using high-speed sorting into RPMI supplemented with 20% FCS. The purity and viability of sorted cells were checked immediately after sorting with PI staining and in all of the experiments exceeded 97%. Cells from 5-10 mice from each group were pooled, and 3x10⁴ cells were transferred in 500 uL DMEM i.v. into mice harboring tumors.

Šustić M., Cokarić Brdovčak M., Lisnić B., Materljan J., Juranić Lisnić V., Rožmanić C., Indenbirken D., Hiršl L., Busch D.H., Brizić I., Krmpotić A, & Jonjić S. (2021). Memory CD8 T Cells Generated by Cytomegalovirus Vaccine Vector Expressing NKG2D Ligand Have Effector-Like Phenotype and Distinct Functional Features. Frontiers in Immunology, 12, 681380.

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Assessing Tumor-Specific CD8+ T Cell Responses

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To estimate in vivo presence of T cells specific to representative peptides from our dataset general ELISPOT assay was performed. Briefly, mice bearing EL4, B16, LLC or MC38 tumors (1.5 cm in diameter) were sacrificed and their spleens were harvested. CD8⁺ T cells were isolated from the spleen using magnetic beads (Miltenyi), mixed with irradiated total splenocytes from the naïve mouse (as the source of antigen-presenting cells) and re-stimulated with 2 μg/ml individual synthetic peptide or 1 μg/ml pool of “PNT-sensitive” or “resistant” peptides (all the peptides used in this study were synthesized by GenScript). Cells were cultured for 48 h in the PVDF plates (Millipore-Sigma) pre-coated with IFN-γ capture antibody (Mabtech), then the plates were washed and developed according to manufacturer procedure (Mabtech). Plates were counted with the use of ImmunoSpot reader (CTL, Cleveland, OH). Values obtained for the cells cultured without peptide restimulation were used as a background and were subtracted from the corresponding values for peptide restimulation.
For isolation of tumor CD8⁺ T cells, tumors were dissociated with Miltenyi tumor dissociation kit with the following isolation of CD8⁺ T cells using magnetic beads (Miltenyi). The purity of isolated splenic and tumor CD8⁺ T cells was >95%.

Tcyganov E.N., Sanseviero E., Marvel D., Beer T., Tang H.Y., Hembach P., Speicher D.W., Zhang Q., Donthireddy L.R., Mostafa A., Tsyganova S., Pisarev V., Laufer T., Ignatov D., Ferrone S., Meyer C., Hajjami H.M., Speiser D.E., Altiok S., Antonia S., Xu X., Xu W., Zheng C., Schuchter L.M., Amaravadi R.K., Mitchell T.C., Karakousis G.C., Yuan Z., Montaner L.J., Celis E, & Gabrilovich D.I. (2022). Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy. Cancer cell, 40(10), 1173-1189.e6.

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Generation of Monocyte-Derived Dendritic Cells for T Cell Activation

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Monocytes were purified, cultured, and matured to monocyte-derived dendritic cells (MD-DCs) as described previously (2 (link)). Briefly, CD14⁺ cells were selected using magnetic beads (Miltenyi Biotec) and cultured for 7 days in the presence of 2 ng/ml rhIL-4 and 10 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (both from Peprotech, Rocky Hill, NJ). MD-DCs were matured by addition of 10 ng/ml recombinant human TNF (eBioscience) on day 6. Matured MD-DCs were pulsed with a 10 μM concentration of the relevant peptide. In parallel, autologous CD8⁺ T cells were purified using magnetic beads (Miltenyi Biotec) and labeled with Pacific Blue succinimidyl ester (PBSE; Life Technologies) as described previously (2 (link)). Subsequently, MD-DCs and CD8⁺ T cells were cocultured in the presence of anti-CD28 MAb (0.5 μg/ml; BD Biosciences) at a ratio of 1:30 (MD-DCs/CD8⁺ T cells). rhIL-2 (20 U/ml) was added on days 4 and 8. On day 12, cells were used for phenotypic and functional analyses.

Nitschke K., Flecken T., Schmidt J., Gostick E., Marget M., Neumann-Haefelin C., Blum H.E., Price D.A, & Thimme R. (2014). Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+ T Cells in Chronic Infection. Journal of Virology, 89(1), 25-34.

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Evaluating CD4+ T Cell Proliferation

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Splenic mononuclear cells were isolated from BALB/c mice, and CD4⁺ T cells were purified by negative selection using magnetic beads and the specific kit, as described by the manufacturer’s instruction (Miltenyi Biotech, Auburn, CA, USA). BMDCs were stimulated with LPS alone or together with ALX at 2 or 10 μM for 48 h; these cells were purified by positive selection using magnetic beads (Miltenyi Biotech) and treated with mitomycin C (30 mg/L, Sangon Biotech, Shanghai, China) for 30 min before harvesting, and washed with PBS twice to terminate the influence of drug on T cell proliferation. The different groups of BMDCs were co-cultured in triplicate with 1 × 10⁵ CD4⁺ T cells at a ratio (DC:T) of 1:5, 1:10, or 1:20, respectively in 96-well plates for 3 days. The CD4⁺ T cells alone served as the control. The proliferation in individual groups of T cells was determined by CCK-8 assay. The proliferation index (PI) was calculated as PI = (co-culture well OD value-medium OD value)/(T cell well OD value-medium OD value) [40 (link)].

Quan M.Y., Song X.J., Liu H.J., Deng X.H., Hou H.Q., Chen L.P., Ma T.Z., Han X., He X.X., Jia Z, & Guo L. (2019). Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses. Journal of Neuroinflammation, 16, 52.

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Lymphocyte Proliferation Dynamics in LCMV Infection

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Naïve B6 mice were infected with 2 × 10⁵ PFU LCMV-Armstrong i.p. on D-15 and D-6. At D0, naïve Thy1.1+ P14 cells were isolated from the spleen via magnetic bead (Miltenyi) sorting on Thy1.1 and labeled with 1uM carboxyfluorescein succinimidyl ester (CFSE) at 37°C for 15 min. 1 × 10⁶ CFSE-labeled naïve P14 cells were adoptively transferred i.v. into D-15 and D-6-infected mice. Three days post-transfer, spleens were harvested to monitor division of labeled P14 cells. Homeostatic proliferation (HP) assays were performed by injecting D99 memory sham and splx mice with 2 mg BrdU i.p. followed by 0.8 mg/ml BrdU in drinking H₂O for 2 weeks. Bone marrow (BM), spleen, lung, and cervical, mediastinal, and inguinal lymph nodes were subsequently harvested, and P14 cells were analyzed for BrdU incorporation.

Kim M.T, & Harty J.T. (2014). Splenectomy Alters Distribution and Turnover but not Numbers or Protective Capacity of de novo Generated Memory CD8 T-Cells. Frontiers in Immunology, 5, 568.

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Treg Suppression Assay for CD4+ T-cells

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Splenocytes were collected from animal cohorts 33 days post-tumor inoculation, and CEA-specific CD4⁺ lymphoproliferation and CD8⁺ T-cell responses were evaluated as previously described (23 (link)). A Treg suppression assay was performed as previously described (21 (link)) with some modification: Tregs were isolated from tumors of control and treated animals via magnetic bead (Miltenyi Biotech, San Diego, CA) and CD4⁺ T-cell proliferation was measured.

Fabian K.P., Malamas A.S., Padget M.R., Solocinski K., Wolfson B., Fujii R., Sater H.A., Schlom J, & Hodge J.W. (2020). Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable. Cancer immunology research, 9(2), 239-252.

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