Anti cd3 microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-CD3 microbeads are a lab equipment product designed for the activation and expansion of T cells. They are composed of magnetic particles coated with anti-CD3 antibodies, which bind to the CD3 receptor on the surface of T cells, triggering their activation.

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Lab products found in correlation

Ls column, by Miltenyi Biotec (3 mentions) Interleukin 2 (il 2), by R&D Systems (2 mentions) Anti cd4 or anti cd8 antibodies, by Thermo Fisher Scientific (2 mentions) Anti mouse cd154 mr1 clone, by BioXCell (2 mentions) Armenian hamster igg isotype, by BioXCell (2 mentions) S3e cell sorter, by Bio-Rad (2 mentions) Macsquant, by Miltenyi Biotec (2 mentions) Anti cd3 antibody, by BioLegend (2 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions) Quadromacs separator, by Miltenyi Biotec (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) Aim 5 medium, by Thermo Fisher Scientific (1 mentions) Anti cd34 antibody, by BioLegend (1 mentions) Cd34 microbead, by Miltenyi Biotec (1 mentions) Anti cd4 antibody, by BioLegend (1 mentions) Anti cd8 antibody, by BioLegend (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Human ab serum, by Merck Group (1 mentions) Anti cd33 microbeads, by Miltenyi Biotec (1 mentions) Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)

27 protocols using anti cd3 microbeads

Enrichment of Human iNKT Cells

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Culture-expanded human iNKT cells were stained with PBS57-CD1d tetramer phycoerythrin (PE) and enriched with anti-PE MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD3+ T cells were isolated from human PBMCs with anti-CD3 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). A QuadroMACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) and LS Columns (Miltenyi Biotec, Bergisch Gladbach, Germany) were used according to the manufacturer’s instructions.

Schmid H., Schneidawind C., Jahnke S., Kettemann F., Secker K.A., Duerr-Stoerzer S., Keppeler H., Kanz L., Savage P.B, & Schneidawind D. (2018). Culture-Expanded Human Invariant Natural Killer T Cells Suppress T-Cell Alloreactivity and Eradicate Leukemia. Frontiers in Immunology, 9, 1817.

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PBMC Isolation and CAR-T Cell Generation

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Density-gradient centrifugation was used to isolate PBMCs from healthy donor samples or patients with MM by Ficoll-Paque (General Electric). T cells were isolated from PBMCs with anti-CD3 microbeads (Miltenyi Biotec, 130-050-101) and were cultured in AIM V Medium (Thermo Fisher Scientific) with 5% human AB serum (MilliporeSigma) and 400 IU IL-2 (R&D Systems). Dynabeads of human T-activator CD3/CD28 (Thermo Fisher Scientific, 11161D) were added for T cell expansion and activation (26 (link)). T cells were activated for 2–3 days prior to transduction.
Lentivirus particles were used to transduce T cells. T cells and concentrated lentiviruses were added into RetroNectin-precoated plates (Takara Bio) (26 (link)). Cells were cultured in AIM V Media for 24 hours, and the transduction step was repeated. After 24 hours, cells were washed with PBS and cultured in fresh media for 7 days. CAR-T cells were detected by BD FACSVerse flow cytometry with anti-CD34 antibodies (BioLegend, clone: 561, no. 343606), anti-CD3 antibodies (BioLegend, clone: HIT3a, no. 300318), and CD34 microbeads (Miltenyi Biotec, 130-046-703) for isolation. CD4 or CD8 phenotypes of CAR-T cells were detected by flow cytometry using anti-CD4 antibodies (BioLegend, clone: RPA-T4, no. 300508) and anti-CD8 antibodies (BioLegend, clone: SK1, no. 344722).

Sun F., Cheng Y., Chen J.R., Wanchai V., Mery D.E., Xu H., Gai D., Al Hadidi S., Schinke C., Thanendrarajan S., Zangari M., van Rhee F., Tricot G., Shaughnessy JD J.r, & Zhan F. (2024). BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions. The Journal of Clinical Investigation, 134(1), e171396.

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Isolation and Characterization of Myeloid-Derived Suppressor Cells

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MDSC (“suppressors”) were enriched using negative selection for HLA-DR (anti-HLA-DR microbeads and LD columns, Miltenyi Biotec, Auburn, CA) and positive selection for CD33 (anti-CD33 microbeads and LS columns, Miltenyi Biotec) from a healthy donor’s fresh PBMC. HLA-DR⁻CD33⁻ cells were further depleted of CD3+ cells (anti-CD3 microbeads and LS columns, Miltenyi Biotec) and used as “non-suppressor controls”. An aliquot of autologous PBMC was set aside for use as the “responder” population. “Responders” were mixed with “suppressors” or “non-suppressor controls” and stimulated with anti-CD3/CD28 beads (1:1, Dynabeads,Thermo Fisher Scientific, Waltham, MA) for 4 days at 37 °C. CD3+ responder proliferation was measured using intracellular Ki67 labeling as described above.

Apodaca M.C., Wright A.E., Riggins A.M., Harris W.P., Yeung R.S., Yu L, & Morishima C. (2019). Characterization of a whole blood assay for quantifying myeloid-derived suppressor cells. Journal for Immunotherapy of Cancer, 7, 230.

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Dissecting T Cell Responses in Murine HSV-1 Infection

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Spleens were harvested and single cell suspensions created by filtration through 70 μm mesh in RPMI1640 culture media containing 10% fetal bovine serum and antibiotics. Splenocytes were pelleted and erythrocytes lysed using 0.84% NH₄Cl (J.T. Baker) in H₂O. For T cell repertoire profiling, splenocytes were labeled with anti-CD45, anti-CD3, and anti-CD8 antibodies (eBioscience) and MHC class I K^b tetramers provided by the NIH Tetramer Core Facility for identification of HSV-1 specific CD8⁺ T cells. Tetramer-labeled cells were analyzed using a MacsQuant flow cytometer (Miltenyi Biotec) as previously described.^{23 (link)} For adoptive transfers into Ifnar1^−/− mice, bulk preparations of CD3⁺ splenocytes were obtained by immunomagnetic isolation using anti-CD3 microbeads (Miltenyi Biotec) according to the manufacturer’s directions. Alternatively, splenocytes were labeled with anti-CD4 or anti-CD8 antibodies (eBioscience) and sorted using an S3e cell sorter (Biorad) for adoptive transfers into TCRα^−/− mice. Adoptive transfer of isolated cells was mediated by intravenous retroorbital injection. In some experiments, TCRα^−/− mice were injected i.p. with 250 μg anti-mouse CD154 (MR1 clone) or Armenian hamster IgG isotype (both from BioXcell) at days 0, 3, and 6 p.i. to evaluate CD40-dependent immune activation following adoptive transfer of CD4 T cells.

Royer D.J., Hendrix J.F., Larabee C.M., Reagan A.M., Sjoelund V.H., Robertson D.M, & Carr D.J. (2019). Vaccine-induced Antibodies Target Sequestered Viral Antigens to Prevent Ocular HSV-1 Pathogenesis, Preserve Vision, and Preempt Productive Neuronal Infection. Mucosal immunology, 12(3), 827-839.

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T Cell Isolation and Activation

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The anti-CD3 microbeads (Miltenyi Biotec, Germany) was used to sort CD3⁺ T cells in mouse spleen. Then, anti-CD3 antibody (2 μg/mL) and anti-CD28 antibody (1 μg/mL) (BioLegend, San Diego, CA, USA) were used to activate T cells.

Zhang S., Fan S., Wang Z., Hou W., Liu T., Yoshida S., Yang S., Zheng H, & Shen Z. (2022). Capecitabine Regulates HSP90AB1 Expression and Induces Apoptosis via Akt/SMARCC1/AP-1/ROS Axis in T Cells. Oxidative Medicine and Cellular Longevity, 2022, 1012509.

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Characterization of MDSC Suppression on T Cell Proliferation

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CD3⁺T cells were isolated from PBMNC of HD by anti-CD3 microbeads (Miltenyi Biotec) and labeled with CellTrace^TM Violet Cell Proliferation kit (5 µM; Invitrogen, Waltham, USA). Isolated MDSC were co-cultured with allogeneic CD3⁺T cells for 72 h at ratios of 1:32, 1:16, 1:8, 1:4, 1:2 or 1:1 in the presence of anti-CD3/anti-CD28 Dynabeads^® (Gibco, Grand Island, USA). Cells were then stained with APC-Cy7-conjugated anti-human CD3, PerCP-Cy5.5-conjugated antihuman CD4 and APC-conjugated anti-human CD8 (Biolegend) antibodies. The proliferation of CD3⁺, CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells was evaluated by flow cytometry.^{14 (link)} For the activation assay, cells were stained with APC-Cy7-con-jugated anti-human CD3, FITC-conjugated anti-human CD69 and PE-conjugated anti-human CD25 (Biolegend) antibodies.

Dong P., Chen L., Wu H., Huo J., Jiang Z., Shao Y., Ren X., Huang J., Li X., Wang M., Nie N., Zhang J., Jin P., Zheng Y, & Ge M. (2022). Impaired immunosuppressive role of myeloid-derived suppressor cells in acquired aplastic anemia. Haematologica, 107(12), 2834-2845.

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Isolation of Intestinal Epithelial Cells

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Human intestinal (jejunal) tissues were obtained from otherwise healthy subjects undergoing gastric bypass for obesity in accordance with IRB approved protocol. IECs were isolated from tissue segments by enzyme digestion, as described previously [28 (link)], and purified by depletion of CD3⁺ T cells using anti-CD3 Microbeads (Miltenyi Biotec).

Shen R., Achenbach J., Shen Y., Palaia J., Rahkola J.T., Nick H.J., Smythies L.E., McConnell M., Fowler M.G., Smith P.D, & Janoff E.N. (2015). Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women. PLoS ONE, 10(12), e0145150.

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Comprehensive Immunophenotypic Analysis of B and T Cells

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PBMCs were enriched for CD19⁺ cells by magnetic cell separation (MACS) according to the manufacturer’s manual (Miltenyi Biotec, Cat#130-050-301). For T cell analysis, PBMCs were enriched by MACS using anti-CD3 microbeads (Miltenyi Biotec, Cat#130-050-101). For FACS analysis and cell sorting, CD19⁺ B cells were incubated with antibodies specific for CD19, CD27, CD38, IgM, IgG, IgD, IgA, and κ light chains. CD3⁺ T cells were FACS-sorted using anti-CD3, anti-CD4, and anti-CD8 antibodies. Bone marrow samples were thawed, washed, and incubated with antibodies specific for CD10, CD34, CD38, CD19, and the IgM µ-chain. After sorting, collected cells were immediately processed or frozen in 90% FBS with 10% DMSO. Cell populations were sorted according to the following phenotypes: 1) naïve B cells: CD19⁺CD27⁻IgM⁺IgD⁺; 2) IgM memory B and Activated IgM cells: CD19⁺CD27⁺IgM⁺; 3) IgG/A⁺ switched memory and Activated IgG/A⁺ cells: CD19⁺CD27⁺IgG/A⁺; 4) IgK⁺ B cells: CD19⁺IgK⁺; 5) IgL⁺ B cells: CD19⁺IgK⁻; 6) CD4 T cells: CD3⁺CD4⁺; 7) CD8 T cells: CD3⁺CD8⁺; 8) pro-B cells: CD38⁺CD19⁺CD10⁺CD34⁺µ-chain⁻; 9) pre-B cells: CD38⁺CD19⁺CD10⁺CD34⁻µ-chain⁻; 10) immature B cells: CD38⁺CD19⁺CD10⁺CD34⁻ µ-chain⁺.

Lebedin M., Foglierini M., Khorkova S., Vázquez García C., Ratswohl C., Davydov A.N., Turchaninova M.A., Daubenberger C., Chudakov D.M., Lanzavecchia A, & de la Rosa K. (2022). Different classes of genomic inserts contribute to human antibody diversity. Proceedings of the National Academy of Sciences of the United States of America, 119(36), e2205470119.

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Engineered nb70CAR-T Cells for Immunotherapy

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T-cells were isolated from commercial peripheral blood mononuclear cell (PBMC) (Milestone® Biotechnologies, China) using anti-CD3 microbeads (Miltenyi, Germany), and were activated by microbeads (Dynabeads™ Human T-Activator CD3/CD28) (Gibco, USA). The CD70 gene was knocked-out by electroporation with Cas9 protein and small guide RNA (Genscript Biotech Corporation, China) using the Celetrix system. Eight hours post electroporation, CD70 knocked-out T-cells (KO T) were transduced with lentivirus encoding nb70CAR. After 24 h, nb70CAR-T and KO T-cells were centrifuged, resuspended in fresh culture medium, and sustained in a concentration of 1 ~ 2 × 10⁶ cells/mL. The complete culture medium was CTS™ OpTmizer™ medium (Gibco, USA) supplemented with 10% FBS and 200 IU/mL rhIL-2 (PeproTech, USA) and 100ug/mL L-glutamine (Gibco, USA).
Lentivirus encoding nbCD70CAR was generated by Lenti-X™293 T cells using psPAX2, p.MD2.G packaging plasmids and transfer-plasmid with Lipo3000 (Invitrogen, USA). Supernatants containing the released virus were collected 48 h post-transfection and concentrated by ultracentrifugation. Lentivirus was stored at – 80 ℃.

Cheng J., Ge T., Zhu X., Wang J., Zeng Y., Mu W., Cai H., Dai Z., Jin J., Yang Y., Hu G., Mao X., Zhou J., Zhu L, & Huang L. (2023). Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia. Cancer Immunology, Immunotherapy, 72(7), 2331-2346.

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Generation of TCR-modified T Cells

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Jurkat 76 was transduced with CD8α and CD8β cDNAs to generate Jurkat 76/CD8 as reported previously18 (link). Jurkat 76 or Jurkat 76/CD8 transfectants were further transduced with individual TCRβ genes along with SIG35α, and the transfectants were purified using anti-CD3 Microbeads (Miltenyi Biotec). K562-based artificial antigen-presenting cells (aAPC) stably express mutated HLA–A2 in conjunction with CD80 and CD8323 (link)26 (link). The mutated HLA–A2 molecules bear two amino acid substitutions at positions 227 and 228 that abrogate the interaction with CD857 (link). aAPC was engineered to constitutively secrete IL-21 to enable T cell expansion26 (link)58 (link). PG13-based packaging cells were generated by first transfecting Phoenix Eco cells with 20 μg of DNA for each construct using the TransIT-293 reagent (Mirus Bio, Madison, WI). PG13 cells were then transduced with supernatant from Phoenix Eco cells. PG13-derived retrovirus supernatant was utilized to transduce TCR genes into Jurkat 76, Jurkat 76/CD8, and human primary T cells as reported previously18 (link). A retroviral vector encoding ΔNGFR alone was employed as a control vector.

Nakatsugawa M., Rahman M.A., Yamashita Y., Ochi T., Wnuk P., Tanaka S., Chamoto K., Kagoya Y., Saso K., Guo T., Anczurowski M., Butler M.O, & Hirano N. (2016). CD4+ and CD8+ TCRβ repertoires possess different potentials to generate extraordinarily high-avidity T cells. Scientific Reports, 6, 23821.

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