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Cd3 28 beads

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CD3/28 beads are a type of magnetic bead coated with antibodies against the CD3 and CD28 molecules on the surface of T cells. These beads are used to activate and expand T cells in vitro for various research and clinical applications.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Facsdiva software, by BD (1 mentions) Retronectin, by Takara Bio (1 mentions) Cellfix, by BD (1 mentions) Resiquimod r848, by InvivoGen (1 mentions) Rlt plus buffer, by Qiagen (1 mentions) Ionomycin iono, by Merck Group (1 mentions) Aim 5, by Thermo Fisher Scientific (1 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions) Cd3 microbeads, by Miltenyi Biotec (1 mentions) Ionomycin, by Merck Group (1 mentions) Golgistop, by BD (1 mentions) Golgiplug, by BD (1 mentions)

Spelling variants of this product

Anti-CD3/28 beads (11 citations) Dynabeads™ Human T‐Activator CD3/28 beads (2 citations) Anti-CD3/28 antibody-coated beads (2 citations)

10 protocols using cd3 28 beads

1

DC-CD4+ T Cell Co-Culture Assay

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DCs were harvested from the wells, and washed two times with PBS. Simultaneously, CD4+ T cells from the same patient were thawed and washed two times with PBS. CD4+ T cells were stained with CellTrace Violet according to the manufacturer’s protocol (Invitrogen, USA). DCs and T cells were combined in a round-bottom 96-wells plate (Corning Costar, Sigma Aldrich) in a ratio of 1:10 in triplo. As positive control, T cells were stimulated with 1:10 CD3/28 beads (Thermo Fisher Scientific) (Figure 1). After 6 days, supernatant was pooled for analysis with ELISA, and T cell polarization (Th1 (CD4+CXCR3+), Th2 (CD4+CRTH2+), and Treg (CD4+CD25+CD127−FoxP3+)) and proliferation were measured with flow cytometry. Flow cytometry data were analyzed with FACS DIVA software (BD).
Hayen S.M., Knulst A.C., Garssen J., Otten H.G, & Willemsen L.E. (2021). Fructo-Oligosaccharides Modify Human DC Maturation and Peanut-Induced Autologous T-Cell Response of Allergic Patients In Vitro. Frontiers in Immunology, 11, 600125.
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2

Cytolytic Activity of CAR T Cells

Cited 4 times
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Frozen human T cells collected from a single health donor were activated with CD3/28 beads (ThermoFisher), expanded in vitro, thawed and electroporated with mRNA transcripts encoding c-Met BBZ CAR, mesothelin BBZ CAR and CD19 BBZ CAR as described [11 (link)]. We have previously demonstrated that the cytotoxic activity of mRNA c-Met-CAR T cells was specific against c-Met expressing cell lines such as M30, a human tumor cell line derived from mesothelioma [17 (link)] and cytotoxic activity was not observed against NCI-H522, a lung cancer cell line which lacks c-Met expression [16 (link)]. To confirm the cytolytic activity on c-Met expressing breast cancer cell lines, BT20 (ATCC HTB-19) and TB129 [14 (link)], tumor cells were loaded with 51Cr and incubated with mRNA electroporated CAR T cells (effector cells or E) at various E:T ratios in V-bottom plates for 4 hours. 51Cr released as a result of cell lysis was quantified as described [18 (link)]. All in vitro cell killing assays were performed in duplicates and repeated in at least three independent experiments.
Tchou J., Zhao Y., Levine B.L., Zhang P.J., Davis M.M., Melenhorst J.J., Kulikovskaya I., Brennan A.L., Liu X., Lacey S.F., Posey A., Williams A.D., So A., Conejo-Garcia J.R., Plesa G., Young R.M., McGettigan S., Campbell J., Pierce R.H., Matro J.M., DeMichele A.M., Clark A.S., Cooper L.J., Schuchter L.M., Vonderheide R.H, & June C.H. (2017). Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) T cells in metastatic breast cancer. Cancer immunology research, 5(12), 1152-1161.
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3

Engineered T-cell receptor expression

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TCR-α and TCR-β gene sequences from selected T cell clones were determined by RT-PCR using multiplex primers as reported.27 (link),28 (link) To increase gene expression, whole TCR sequences were codon optimized, and the constant regions were murinized as previously reported.29 (link) The TCRα and TCRβ genes were cloned into the retroviral vector, pSplice-TCR-BC1 (Takara Bio).30 (link) The vector was transduced into the GP13 packaging cell line (ATCC) and the culture supernatant was collected after 48 h, and infected to 3-day activated T cells with CD3/28 beads (Thermo Fisher Scientific) in the presence of 50 U/mL IL-2 in a Retronectin (Takara Bio)-coated plate by “Spinfection” according to the manufacturer’s instruction. Resultant TCR-T cells were analyzed for transduction efficiency five days after transduction with PE-labeled anti-TCRVβ18 antibody (#IM2049, Beckman Coulter, USA).
Katsuyama N., Kawase T., Barakat C., Mizuno S., Tomita A., Ozeki K., Nishio N., Sato Y., Kajiya R., Shiraishi K., Takahashi Y., Ichinohe T., Nishikawa H, & Akatsuka Y. (2023). T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation. Nagoya Journal of Medical Science, 85(4), 779-796.
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4

NK Cell Activation in HIV-1 Infection

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Levels of NK cell activation have been determined through expression of CD107a on the surface of NK cells (Alter et al., 2004 (link)). Enriched primary NK cells were cultured for 3 days in complete media supplemented with 50 IU/ml hrIL-2 and 1 ng/ml hrIL-15 and subsequently co-cultured with differentially stimulated autologous CD4+ T cells. Autologous CD4+ T cells were either cultured with CD3/28 beads (Gibco) or PHA with or without subsequent HIV-1 infection for a total of 3 days. Monensin was added one hour after setup of the co-culture followed by additional 3 hours of incubation. Cells were stained for viability (Live/Dead Blue), expression of CD3, CD4, CD16, CD56 and then fixed with paraformaldehyde (Cell fix, BD).
He X., Simoneau C.R., Granoff M.E., Lunemann S., Dugast A.S., Shao Y., Altfeld M, & Körner C. (2016). Assessment of the antiviral capacity of primary natural killer cells by optimized in vitro quantification of HIV-1 replication. Journal of immunological methods, 434, 53-60.
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5

Stimulation and Analysis of Immune Cell Subsets

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Whole PBMC were left untreated or stimulated with 100 ng/ml staphylococcal enterotoxin B (SEB, Sigma) at 2*10e6 cells per ml for 18 h at 37 °C. Supernatants were harvested and stored at −80 °C until use. CD4+, CD8+, CD14+, CD19+, CD56+ sorted cells, and moDCs were either left untreated or stimulated at 2*10e6 cells per ml with 100 ng/ml SEB, 1 μg/ml resiquimod (R848) (InvivoGen)—a toll-like receptor 7 and 8 ligand, a potent activator of both monocytes and B cells—, 10 μ/ml CD3/28 beads (Gibco, Life Technologies) for 18 h or 100 ng/ml phorbol myristate acetate (PMA, Sigma) and 1 μg/ml ionomycin (iono, Sigma) for 6 h at 37 °C.
For mRNA analysis, cells were lysed with RLT Plus buffer (Qiagen) and kept at −20 °C until further extraction.
Perriard G., Mathias A., Enz L., Canales M., Schluep M., Gentner M., Schaeren-Wiemers N, & Du Pasquier R.A. (2015). Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes. Journal of Neuroinflammation, 12, 119.
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6

Reprogramming T Cells into iPSCs

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CD3+CD45+side scatter low T cells were isolated by
FACS and stimulated with CD3/28 beads (Life Technologies) and transduced
with reprogramming factors via Sendai virus vectors as previously described
(Nishimura et al., 2013 (link)). As
noted, transduced cells were seeded onto murine embryonic fibroblasts (MEF)
feeder cells and cultured in T cell medium (RPMI-1640 supplemented with
10% human AB Serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100
ng/ml streptomycin), which was gradually replaced with human iPSC medium
(Dulbecco’s modified Eagle’s medium/F12 supplemented with
20% knockout serum replacement, 2 mM L-glutamine, 1%
nonessential amino acids, 10 mM 2-mercaptoethanol, and 5 ng/ml basic
fibroblast growth factor). Formed iPSC colonies were picked up and
maintained in Essential 8 medium.
Chao M.P., Gentles A.J., Chatterjee S., Lan F., Reinisch A., Corces M.R., Xavy S., Shen J., Haag D., Chanda S., Sinha R., Morganti R.M., Nishimura T., Ameen M., Wu H., Wernig M., Wu J.C, & Majeti R. (2017). Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease. Cell stem cell, 20(3), 329-344.e7.
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7

Multiplex Cytokine Profiling of Activated DCs and T Cells

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50,000 sorted DCs (2.5 ×105 cells /ml) were activated with different stimuli and culture supernatants from 24-h were analyzed for cytokine production. 500,000 T cells (1 ×106 cells /ml) were stimulated with CD3/28-beads (Life Technology) at 1:1 ratio and the supernatants were harvested at 6-h for T cell cytokine measurement. DC and T cell cytokines were measured using the multiplex kit (EMD Millipore, Billerica, MA) as per the manufacturer's protocol and analyzed using the BioPlex Luminex 100 instrument (EMD Millipore). Cytokine concentrations were calculated using Milliplex analyst software with a 5-parameter curve-fitting algorithm applied for standard curve calculations.
Yu C.I., Becker C., Metang P., Marches F., Wang Y., Toshiyuki H., Banchereau J., Merad M, & Palucka K. (2014). Human CD141+ DCs induce CD4+ T cells to produce type 2 cytokines. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4335-4343.
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8

Isolation and Characterization of CD3+ T Cells

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Peripheral blood mononuclear cells (PBMNCs) were isolated through centrifugation using Ficoll reagent (Haoyang, China). Then CD3 + T cells were positive selected by CD3 microbeads as we previously reported (Miltenyi Biotec, Auburn, USA). For flow cytometry analysis, cells were incubated with fluorochrome-labeled Abs: Fitc CD4, APC CD8, APC/CY7 interferon-γ(IFN-γ), PE interleukin-4 (IL-4) and APC interleukin-17A (IL-17A). The CD3 + T cells were seeded onto 48-well culture plates at a density of 5 * 105 cells/well and cultured with AA/HD MSC-derived exosomes in 500 μl AIM V (Gibco) medium, 5 μl CD3/28beads (Invitrogen, Waltham, USA) were added, 1 μl Cell Stimulation Cocktail (Invitrogen, Waltham, USA) which contained PMA, ionomycin, brefeldin A and monensin was added for 6 h at 37 °C in 5% CO2 before harvest. Then cells were collected to detect intracellular cytokine as described previously. The antibodies involved in this study are listed in Additional file 5: Table S2.
Wang S., Huo J., Liu Y., Chen L., Ren X., Li X., Wang M., Jin P., Huang J., Nie N., Zhang J., Shao Y., Ge M, & Zheng Y. (2023). Impaired immunosuppressive effect of bone marrow mesenchymal stem cell-derived exosomes on T cells in aplastic anemia. Stem Cell Research & Therapy, 14, 285.
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9

Conventional T cell proliferation assay

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Conventional T cells (Tcon cells, CD4+CD25−) were stained with CellTrace Violet (Invitrogen, Carlsbad, CA) following manufacturer's recommendations and stimulated with 1:1 CD3/28 beads (Invitrogen, Carlsbad, CA) at 50,000 cells/well of a 96-well plate, in presence of Tregs at a different ratios; and then incubated for three days at 37°C followed by assessment of Tcon-proliferation using LSR Fortessa and analyzed using FlowJo software.
Kellner J.N., Delemarre E.M., Yvon E., Nierkens S., Boelens J.J., McNiece I., Olson A., Nieto Y., Ciurea S., Popat U., Ahmed S., Champlin R., Ramos J., Nishimoto M., Ma H., Ke Z., Thall P., Khoury J.D., Negrin R., Andersson B, & Parmar S. (2018). Third party, umbilical cord blood derived regulatory T-cells for prevention of graft versus host disease in allogeneic hematopoietic stem cell transplantation: feasibility, safety and immune reconstitution. Oncotarget, 9(86), 35611-35622.
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10

T Cell Activation and Cytokine Analysis

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In vitro derived T cells from hESCs and hiPSCs and primary T cells from human PBMC were stimulated by incubation with CD3/28 beads (Invitrogen) for 3 days before analysis by flow cytometry. For cytokines production assays, T cells were stimulated with 10 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) for 12 hours. GolgiStop and GolgiPlug (BD Bioscience) were also added at the same time.
Chang C.W., Lai Y.S., Lamb LS J.r, & Townes T.M. (2014). Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells. PLoS ONE, 9(5), e97335.
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