Soluble anti cd28 clone cd28

Manufactured by BD

Sourced in United States

Soluble anti-CD28 (clone CD28.2) is a monoclonal antibody that binds to the CD28 receptor on the surface of T cells. CD28 is a co-stimulatory molecule that plays a crucial role in T cell activation and proliferation. This soluble anti-CD28 antibody can be used in various in vitro applications to study T cell biology and function.

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Lab products found in correlation

Sodium pyruvate, by Lonza (3 mentions) Ack buffer, by Thermo Fisher Scientific (2 mentions) Dynabeads flowcomp human cd3 kit, by Thermo Fisher Scientific (2 mentions) Anti cd3 clone ucht1, by BD (2 mentions) Anti cd3 clone hit3a, by BD (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Sb431542, by Merck Group (1 mentions) Tgf β1, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) Bms 345541, by Merck Group (1 mentions) Ficoll hypaque technique, by Cytiva (1 mentions) Pcr mycoplasma detection kit, by Applied Biological Materials (1 mentions) Rpmi 1640 medium, by Lonza (1 mentions) Jeko 1, by American Type Culture Collection (1 mentions) Maver 1, by American Type Culture Collection (1 mentions) Ficoll hypaque, by Cytiva (1 mentions) Non essential amino acids (neaa), by Lonza (1 mentions) Facsaria fusion cell sorter, by BD (1 mentions) Anti cd3 clone okt3, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Soluble anti-CD28 (clone 37.51, Soluble anti-CD28 mAb (clone 37.51; Soluble anti-CD28 Anti-CD28 (clone CD28.2, Clone CD28.2 Anti-CD28

5 protocols using soluble anti cd28 clone cd28

TGF-β-Induced T Cell Activation Assay

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PBLs from patients with SSc and healthy donors were cultured for 5 days on plates previously coated with anti-CD3 (clone HIT3a, 2 μg/ml; BD Pharmingen, San Diego, CA, USA) antibody at the concentration of 0.5 × 10⁶ cells/ml in complete RPMI 1640 medium supplemented with soluble anti-CD28 (clone CD28.2, 1 μg/ml; BD Pharmingen) antibody and IL-2 (20 U/ml; PeproTech, Rocky Hill, NJ, USA). At the sixth day, cells were stimulated for the last 4 h with a combination of phorbol 12-myristate 13-acetate (10 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) and ionomycin (1 μM; Sigma-Aldrich) in the presence or absence of the optimal concentration of 5 ng/ml TGF-β1 (PeproTech), named TGF-β hereafter, in 0.5 % FCS-containing RPMI 1640 medium with or without a 1-h preincubation with specific inhibitors (SB431542 from Sigma-Aldrich; SB203580 or SIS3 from Calbiochem, San Diego, CA, USA). Jurkat T cells were cultured in 0.5 % FCS-containing medium for 16 h before addition of 5 ng/ml TGF-β for 30 min or 4 h.

Baraut J., Farge D., Jean-Louis F., Masse I., Grigore E.I., Arruda L.C., Lamartine J., Verrecchia F, & Michel L. (2015). Transforming growth factor-β increases interleukin-13 synthesis via GATA-3 transcription factor in T-lymphocytes from patients with systemic sclerosis. Arthritis Research & Therapy, 17(1), 196.

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CLL Peripheral Blood T Cell Activation

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Following approval by the Institutional Review Board and provision of written consent, peripheral blood was obtained from CLL patients treated at Oregon Health and Science University. Peripheral blood mononuclear cells were isolated using standard Ficoll-Hypaque technique (Amersham). Red blood cells were lysed using ACK buffer (Thermo Fisher Scientific). Where indicated, T cells were enriched using Dynabeads FlowComp Human CD3 Kit (Thermo Fisher Scientific). Primary cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2mM L-glutamine, 25 mM HEPES, 100 μM nonessential amino acids, and 1 mM sodium pyruvate (Lonza). T cells were activated using 0.5 μg/mL plate-bound anti-CD3 (clone UCHT1) and 0.5 μg/mL soluble anti-CD28 (clone CD28.2) (BD Biosciences). Mouse fibroblast cell line engineered to express CD40L (L4.5), OCI-LY3, and OCI-LY19 cells were obtained from DSMZ. Pevonedistat (TAK-924) was provided by Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited. BMS-345541 was purchased from Sigma-Aldrich.

Best S., Lam V., Liu T., Bruss N., Kittai A., Danilova O.V., Murray S., Berger A., Pennock N.D., Lind E.F, & Danilov A.V. (2020). Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells. Leukemia, 35(1), 156-168.

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Isolation and culture of cancer cell lines and T cells

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A20, JeKo-1, Mino, Val, Maver-1 and Lewis lung carcinoma (LLC) cells were obtained from American Type Culture Collection (ATCC, USA). Mycoplasma testing was performed regularly (every 2 months) using Mycoplasma PCR detection kit (ABM, Canada). The number of passages between thawing and use in the described experiments ranged between two and five. Cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, 25 mM HEPES, 100 μM nonessential amino acids, and 1 mM sodium pyruvate (Lonza).
Total CD3⁺ T cells were isolated from PBMCs derived from CLL patients by positive selection kit (Invitrogen, USA) and cultured with 0.5 μg/mL plate-bound anti-CD3 (clone UCHT1) and 0.5 μg/mL soluble anti-CD28 (clone CD28.2; BD Biosciences).
Pevonedistat (TAK924) was provided by Takeda Development Center Americas, Inc.

Wang X., Chen C., Vuong D., Rodriguez-Rodriguez S., Lam V., Roleder C., Wang J.H., Kambhampati S., Berger A., Pennock N., Torka P., Hernandez-Ilizaliturri F., Siddiqi T., Wang L., Xia Z, & Danilov A.V. (2023). Pharmacologic targeting of Nedd8-activating enzyme reinvigorates T-cell responses in lymphoid neoplasia. Leukemia, 37(6), 1324-1335.

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Culturing Primary CLL T Cells

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Following approval by the Institutional Review Board and provision of written consent, peripheral blood was obtained from previously untreated CLL patients treated at the City of Hope National Medical Center (IRB#18067). Peripheral blood mononuclear cells (PBMCs) were isolated using standard Ficoll-Hypaque technique (Amersham). Red blood cells were lysed using ACK buffer (Thermo Fisher Scientific). T cells were enriched using Dynabeads FlowComp Human CD3 Kit (Thermo Fisher Scientific). Primary cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 25 mM HEPES, 100 μM non-essential amino acids and 1 mM sodium pyruvate (Lonza). T cells were activated using 0.5 μg/mL plate-bound anti-CD3 (clone UCHT1) and 0.5 μg/mL soluble anti-CD28 (clone CD28.2) (BD Biosciences) in the presence or absence of anti-human IFNAR2 antibody (clone MMHAR-2) from PBL Assay Science (2 μg/ml). Mouse fibroblast cell line engineered to express CD40L (L4.5), OCI-LY3 and U2932 cells were obtained from DSMZ. All cell lines were tested for Mycoplasma at least every 2 months and used after <10 passages. TAK-981 was provided by Takeda Development Center Americas Inc. (Lexington, MA), prepared as described in reference (10 (link)).

Arai H., Tsou C.L, & Charo I.F. (1997). Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors. Proceedings of the National Academy of Sciences of the United States of America, 94(26), 14495-14499.

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Isolation and Stimulation of Human CD4+ T Cell Subsets

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Peripheral blood mononuclear cells (PBMCs) from lupus patients and from volunteers and anonymous donors of Etablissement Français du Sang were isolated from heparinized venous blood by Ficoll density gradient centrifugation (GE Healthcare). CD4⁺ T cells from healthy donors were negatively selected using the MojoSort Human CD4 T Cell Isolation Kit (Biolegend) according to the manufacturer’s instructions. The purity of the CD4⁺ population was typically ≥ 90%. Isolated CD4⁺ T cells were incubated with the following conjugated mAbs: anti-CD4-allophycocyanin (APC) Violet-770 (clone REA 623, Miltenyi); anti-CD45RA-phycoerythrin (PE) Violet-615 (clone 562, Miltenyi); anti-CD25-APC (clone REA 570, Miltenyi), and naive CD4⁺ T cells and Treg subpopulations (aTregs and rTregs) were isolated by a FACSAria™ Fusion cell sorter (BD). Purified naive CD4⁺ T cells, aTregs and rTregs were cultured in complete medium (RPMI 1640 containing 10% FCS, 10 µg/ml gentamicin, and 10 mM HEPES) and plated at 5.10⁵ cells per ml at 37°C. Cells were stimulated with 5 µg/ml plate-bound anti-CD3 (clone OKT3, eBioscience) and with 5 µg/ml soluble anti-CD28 (clone CD28.2, BD Pharmingen). Samples were cultured for 4 hours or 48 hours, harvested, and used for RNA isolation or flow cytometric analysis respectively.

Aubergeon L., Sawaf M., Felten R., Gottenberg J.E., Dumortier H, & Monneaux F. (2021). High BTLA Expression Likely Contributes to Contraction of the Regulatory T Cell Subset in Lupus Disease. Frontiers in Immunology, 12, 767099.

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