Anti cd3 28 beads

Manufactured by Thermo Fisher Scientific

Sourced in Germany

Anti-CD3/28 beads are a type of magnetic beads coated with antibodies against the CD3 and CD28 markers found on the surface of T cells. These beads are designed to activate and expand T cells in vitro for various research and therapeutic applications.

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11 protocols using anti cd3 28 beads

Itpkb-Dependent T and B Cell Activation Assays

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Spleens were removed from Itpkb^+/+ and Itpkb^fl/fl mice. Following RBC lysis, CD4⁺ T cells were purified by negative selection (Miltenyi Biotec) and resuspended in RPMI complete media containing 10% FCS, 2mM L-Glutamine, 100U penicillin, 100 μg/ml streptomycin, 10mM Hepes, 1x NEAA, 1mM Na Pyruvate and 50 μM 2-ME. 5x10⁴ purified CD4⁺ T cells were plated in a 96-well round bottom plate and stimulated with anti-CD3/28 beads (Invitrogen) at either a 0.5:1 or 2.5:1 beads-to-cell ratio. Cells were incubated for 48 h at 37°C, before adding 1μCi/well of ³H-thymidine for an additional 18 h at 37°C. 1x10⁵ B cells purified by negative selection (Miltenyi Biotec) were stimulated with anti-IgM (Fab’2), anti-CD40, or LPS at the concentrations indicated in the presence of rIL-4 at 50ng/mL for 72h at 37°C, before adding Cell Titer Glo (Promega). Luminescence was measured on an Acquest fluorescence reader (Molecular Devices).

Miller A.T., Dahlberg C., Sandberg M.L., Wen B.G., Beisner D.R., Hoerter J.A., Parker A., Schmedt C., Stinson M., Avis J., Cienfuegos C., McPate M., Tranter P., Gosling M., Groot-Kormelink P.J., Dawson J., Pan S., Tian S.S., Seidel H.M, & Cooke M.P. (2015). Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease. PLoS ONE, 10(6), e0131071.

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Isolating and Infecting Primary CD4+ T Cells

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Leukocytes were isolated by density gradient separation from peripheral blood freshly-drawn from healthy donors and either used immediately for cytometry, lysed and frozen for immunoprecipitation/mass spectrometry, or selected for HIV infection using anti-CD4 mAb-based magnetic selection (EasySep) to attain a purity exceeding 95% CD4+ with less than 1% CD8+ cells as assessed by cytometry staining with L120-PE and SK1-FITC (both Becton Dickinson). CD4+ cell preparations were expanded for 3-5 days in RPMI supplemented with 100 U/ml IL-2 (Peprotech), anti-CD3/28 beads (Invitrogen), FBS (Lonza) and PSG (Invitrogen) before infection with the HIV strain NL4-3 [36 (link)]. NL4-3 was generated by infectious molecular clone transfection of HEK293T, with the infectious titre of culture supernatants determined using TZM-Bl [37 (link)]. Infection was performed by incubation of cells with virus at a nominal multiplicity of infection of 0.1 for 4-6hrs at 37°C. Cells were then washed and cultured in RPMI with IL-2 for a further 6 or 7 days before analysis by cytometry.

Apps R., Meng Z., Del Prete G.Q., Lifson J.D., Zhou M, & Carrington M. (2015). Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3594-3600.

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Measurement of Acid Sphingomyelinase Activity

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Enzyme activity of ASM was measured as previously described with minor modifications.^{38 (link), 39 (link)} Briefly, 1 × 10⁶ CD4⁺ T cells were stimulated with anti-CD3/28 beads (the ratio of bead and cell is 1 : 1; Invitrogen) for the indicated times. For single anti-CD3 or CD28 antibody stimulation, mouse anti-human CD3 or CD28 antibody (10 μg/ml; BD Pharmingen) was mixed with crosslinking antibody (10 μg/ml; BD Pharmingen) at 4 °C for 15 min, and then was introduced into CD4⁺ T cells. Stimulation was stopped by putting samples on dry ice for 10 s, and the cells were lysed in sodium acetate buffer (50 mM sodium acetate, pH 5.0, 0.5% NP-40).
Ceramide as the lipid cleavage product of ASM was then measured by TLC. Briefly, 10 μg of cell lysates were incubated with sphingomyelin (0.2 mg, dissolved in a mixture of chloroform and methanol (1 : 1)) for 2 h and then loaded onto a TLC plate, followed by chromatographic separation using a solvent system of chloroform and methanol (3 : 2). Ceramide production was then visualized in iodine vapor, identified using a standard run-in parallel, and scanned by a scanner. Relative ceramide levels were evaluated by Image J software (NIH).

Bai A., Kokkotou E., Zheng Y, & Robson S.C. (2015). Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses. Cell Death & Disease, 6(7), e1828-.

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T-cell Polarization and Activation Protocol

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Human whole blood were obtained from healthy volunteers with written informed consent using protocols approved by Chesapeake Institutional Review Board.
PBMCs, isolated from whole blood using Ficoll centrifugation, were activated with anti-CD3/28 beads (Life Technologies) at 1:1 ratio and polarized into Type 17 cells with human IL-1β (10 ng/mL), IL-6 (10 ng/mL), and IL-23 (10 ng/mL). In some experiments, human recombinant TGFβ (0.5 ng/mL) were added to induce FOXP3 and CD73 expression. After 5 days, cytokine levels in the media were determined using a luminex panel (R&D Systems). Cells were collected for flow cytometry analysis.
PBMCs from cancer patients were purchased from Conversant Bio and activated with anti-CD3/28 beads and differentiated as described above.

Hu X., Liu X., Moisan J., Wang Y., Lesch C.A., Spooner C., Morgan R.W., Zawidzka E.M., Mertz D., Bousley D., Majchrzak K., Kryczek I., Taylor C., Van Huis C., Skalitzky D., Hurd A., Aicher T.D., Toogood P.L., Glick G.D., Paulos C.M., Zou W, & Carter L.L. (2016). Synthetic RORγ agonists regulate multiple pathways to enhance antitumor immunity. Oncoimmunology, 5(12), e1254854.

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Co-Culture of OT-1 T Cells

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Equal numbers of OT-1 (CD45.1) and OT-1 x Rag2^−/− (CD45.2) T cells were co-cultured in complete RPMI + 10% DBS with 1:1000 2-mercaptoethanool (Sigma) containing 1 μM SIINFEKL peptide or anti-CD3/28 beads (Lifetechnologies).

Karo J.M., Schatz D.G, & Sun J.C. (2014). The RAG recombinase dictates functional heterogeneity and cellular fitness in natural killer cells. Cell, 159(1), 94-107.

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Isolating and Expanding Human Thymic Regulatory T Cells

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Thymic (t)Treg were sort-purified from peripheral blood mononuclear cells by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) isolated from human apheresis products (Memorial Blood Center, St. Paul, MN) in a two-step procedure whereby CD25⁺ cells (including tTreg) were positively selected using GMP-grade mAb-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) and then sorted for naïve tTreg (CD4⁺25⁺⁺127^-45RA⁺). Purified cells were stimulated with GMP-quality artificial antigen presenting cells (aAPC) and cultured for 14 days in high dose IL-2 (300 U/ml) as previously reported^{46 (link)}. Expanded tTreg were banked (frozen) on day 14. For the experiments described herein, tTreg were thawed and re-stimulated with GMP-compliant anti-CD3/28 beads (Thermo-Fisher Scientific, Waltham, MA) for 7–10 days. tTreg purity and in vitro suppressive function were assessed at the end of the culture. The tTreg are simply indicated as Treg in the text.

Fiore A., Ugel S., De Sanctis F., Sandri S., Fracasso G., Trovato R., Sartoris S., Solito S., Mandruzzato S., Vascotto F., Hippen K.L., Mondanelli G., Grohmann U., Piro G., Carbone C., Melisi D., Lawlor R.T., Scarpa A., Lamolinara A., Iezzi M., Fassan M., Bicciato S., Blazar B.R., Sahin U., Murray P.J, & Bronte V. (2018). Induction of immunosuppressive functions and NF-κB by FLIP in monocytes. Nature Communications, 9, 5193.

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Evaluating DS-2741a Immunomodulatory Effects

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Human PBMCs were purchased from Cellular Technology Ltd. (OH, USA). Mouse CD3⁺ T cells were isolated from mouse spleen by using EasySep mouse T‐cell isolation kit (STEMCELL, Vancouver, Canada) according to the manufacturer's protocol.
Jurkat cells, Human PBMCs, or CD3⁺ T cells of hu‐ORAI1 KI mouse were cultured in 96‐wells microtiter plates containing the indicated concentrations of DS‐2741a or the isotype control IgG₁ (Eureka Therapeutics, Inc) diluted in RPMI culture media, and were incubated for 1 hour at 37°C in a humidified incubator containing 5% CO₂. The cells were stimulated by 61.7 nmol/L PMA (Phorbol 12‐myristate 13‐acetate, SIGMA) and 141 nmol/L A23187 (SIGMA) or anti‐CD3/28 beads (Thermo Fisher Scientific K.K.) at a bead‐to‐cell ratio of 1:1 for 18 to 24 hours at 37°C in a humidified incubator containing 5% CO₂. The supernatants were then collected, and IL‐2 concentration was determined using an ELISA kit (R&D systems, Minneapolis, MN, USA).

Aki A., Tanaka K., Nagaoka N., Kimura T., Baba D., Onodera Y., Wada T., Maeda H., Nakanishi T., Agatsuma T, & Komai T. (2020). Anti‐ORAI1 antibody DS‐2741a, a specific CRAC channel blocker, shows ideal therapeutic profiles for allergic disease via suppression of aberrant T‐cell and mast cell activation. FASEB BioAdvances, 2(8), 478-488.

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Suppression of T Cell Proliferation by Macrophages

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For the suppression of T cells, 1 × 10⁵ CD4 cells from splenocytes of BALB/c mice isolated by magnetic cell sorting (no. 130-045-101, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were seeded and stimulated with anti-CD3/28 beads (no. 11131D, Thermo Fisher Scientific Inc., Waltham, MA) and added to 5 × 10⁴ BMDMs or sorted TAMs per well in a 96-well plate. After 24 h, the proliferation of T cells was analyzed by the cell proliferation assay.

Lee C.C., Lin J.C., Hwang W.L., Kuo Y.J., Chen H.K., Tai S.K., Lin C.C, & Yang M.H. (2018). Macrophage-secreted interleukin-35 regulates cancer cell plasticity to facilitate metastatic colonization. Nature Communications, 9, 3763.

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T cell Differentiation and Cytokine Inhibition

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Human PBMCs were purchased from Cellular Technology Ltd.. Naïve CD4⁺ T cells were isolated from PBMCs using a human Naïve CD4 isolation kit (Miltenyi Biotec K.K., Tokyo, Japan) according to the manufacturer's instructions. To initiate differentiation into Th1, Th2, Th22, and Treg cells, the naïve CD4⁺ T cells (5 × 10⁴/well) were treated with anti‐CD3/28 beads (Thermo Fisher Scientific K.K.) and relevant cytokines (listed below) for 5 days. DS‐2741a or FK‐506 was added 1 hour before the differentiation procedure commenced.
Th1: 5 ng/mL IL‐2 (Peprotech Inc., NJ, USA) and 10 ng/mL IL‐12 p70 (Peprotech Inc.)
Th2: 10 ng/mL IL‐4 (Peprotech Inc.)
Th22: 100 ng/mL TNFα (R&D systems) and 20 ng/mL IL‐6 (Peprotech Inc.)
Treg: 20 ng/mL IL‐2 and 10 ng/mL TGFβ (R&D systems).
To evaluate the inhibitory effects on cytokine production using DS‐2741a or FK‐506, the levels of several cytokines (IFNγ, IL‐13, IL‐31, and IL‐22) were measured using ELISA after treatment with 61.7 nmol/L PMA and 141 nmol/L A23187 for 24 hours (IFNγ, PerkinElmer, Inc. MA, USA; IL‐13 and IL‐31, Thermo Fisher Scientific K.K.; IL‐22, R&D systems). For the evaluation of Treg differentiation, the number of Foxp3⁺CD4⁺ cells was determined by flow cytometry.

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Comprehensive T Cell Proliferation Assay

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For CFSE proliferation assays, ATO-derived CD8SP or CD4SP T cells were isolated by negative selection MACS as above (with further FACS purification of CD4SP T cells as described above) and labeled with 5 μM CFSE (Biolegend, San Diego, CA). Labeled cells were incubated with anti-CD3/CD28 beads (ThermoFisher Scientific, Grand Island, NY) in AIM V/5% human AB serum with 20 ng/ml rh IL-2 (Peprotech, Rocky Hill, NJ), co-stained for CD25 or 4-1BB (Biolegend, San Diego, CA) and analyzed by flow cytometry on day 5. In some experiments CFSE was substituted for CellTrace Violet (CTV; ThermoFisher) with labeling per the manufacturer’s protocol. For in vitro cell expansion assays, 5×10³–1×10⁴ ATO-derived CD8SP or CD4SP T cells isolated as above were plated in 96-well U-bottom plates in 200 μl, and activated/expanded with anti-CD3/28 beads and either 20 ng/mL IL-2 or 5 ng/mL IL-7 and 5 ng/mL IL-15 (Peprotech). Beads were removed on day 4, and fresh medium and cytokines were added every 2–3 days with replating into larger wells as needed. Cells were counted weekly with a hemacytometer. In some experiments, cells were restimulated with fresh anti-CD3/CD28 beads on day 14.

Seet C.S., He C., Bethune M.T., Li S., Chick B., Gschweng E.H., Zhu Y., Kim K., Kohn D.B., Baltimore D., Crooks G.M, & Montel-Hagen A. (2017). Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids. Nature methods, 14(5), 521-530.

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