Anti human cd28 antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-human CD28 antibodies are laboratory reagents used to detect and study the CD28 protein, which is expressed on the surface of T cells. These antibodies can be used in various immunological techniques to identify and isolate CD28-positive cells.

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Lab products found in correlation

Anti human cd3 antibody, by Thermo Fisher Scientific (10 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions) Recombinant human il 2, by R&D Systems (4 mentions) Penicillin and streptomycin, by Solarbio (3 mentions) Ficoll hypaque, by Cytiva (2 mentions) Ms column, by Miltenyi Biotec (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (2 mentions) Ficoll hypaque centrifugation, by Cytiva (2 mentions) Anti human pd 1 antibody, by BioLegend (1 mentions) Recombinant human interleukin 2 il 2, by R&D Systems (1 mentions) Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions) Interleukin 2 (il 2), by BD (1 mentions) Software, by FlowJo (1 mentions) Anti cd4 coated magnetic beads, by Miltenyi Biotec (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Penicillin, by Solarbio (1 mentions) Streptomycin, by Solarbio (1 mentions) Retronectin, by Takara Bio (1 mentions) Ficoll paque, by GE Healthcare (1 mentions)

10 protocols using anti human cd28 antibody

Decitabine Modulates CD8+ T Cells in ITP

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PBMCs were isolated from peripheral venous blood of ITP patients and healthy controls with Ficoll–Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ, USA). CD8⁺ T cells were sorted from PBMCs by magnetic beads and MS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) with a purity of over 90%.
For in vitro assays, CD8⁺ T cells were cultured in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Solarbio, Beijing, China), and were stimulated with recombinant human IL-2 (5 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-human CD3 antibodies (1 ng/mL, eBioscience, San Diego, CA, USA), and anti-human CD28 antibodies (1 ng/mL, eBioscience) for 72 h (37°C, 5% CO₂). Recombinant fusion protein PD-L1-Fc (0.5 μg/mL, R&D Systems), decitabine (100 nM), anti-human PD-1 antibody (10 μg/mL, BioLegend, CA, USA), or PBS were added to the incubation media. Subsequently, CD8⁺ T cells were collected for flow cytometry, CTLs-induced platelet apoptosis, and bisulfite sequence PCR.
For ITP patients receiving decitabine, we isolated CD8⁺ T cells before (0) and after (12 weeks) the administration of decitabine and determine the function of CD8⁺ T cells by flow cytometry and CTLs-induced platelet apoptosis analysis.

Han P., Yu T., Hou Y., Zhao Y., Liu Y., Sun Y., Wang H., Xu P., Li G., Sun T., Hu X., Liu X., Li L., Peng J., Zhou H, & Hou M. (2021). Low-Dose Decitabine Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PD-1 Methylation in Immune Thrombocytopenia. Frontiers in Immunology, 12, 630693.

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PBMC Isolation and CD4+ T Cell Activation

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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of patients with ITP and healthy control subjects with Ficoll-Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ). CD4⁺ T cells were sorted by using magnetic beads and MS Columns (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated cells was >90% as measured by flow cytometry.
PBMCs or sorted CD4⁺ T cells were cultured in RPMI 1640 medium (Life Technologies, Paisley, United Kingdom) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and 1% penicillin and streptomycin (Solarbio, Beijing, China). Cells were stimulated with recombinant human interleukin-2 (IL-2; 5 ng/mL; R&D Systems, Minneapolis, MN), anti-human CD3 antibodies (1 ng/mL; eBioscience, San Diego, CA), and anti-human CD28 antibodies (1 ng/mL; eBioscience) and incubated for 72 hours (37°C, 5% carbon dioxide).^{24 (link)}

Han P., Hou Y., Zhao Y., Liu Y., Yu T., Sun Y., Wang H., Xu P., Li G., Sun T., Hu X., Liu X., Li L., Peng J., Zhou H, & Hou M. (2021). Low-dose decitabine modulates T-cell homeostasis and restores immune tolerance in immune thrombocytopenia. Blood, 138(8), 674-688.

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Regulatory T Cells Modulate Effector T Cell Proliferation

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Fresh CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ Tregs were isolated from PBMCs using a CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec) per manufacturer instructions. CD4⁺CD25⁻ T cells (effector T cells) were labelled with CFSE (5 µmol/L; Sigma‐Aldrich), seeded at 2 × 10⁵ cells/well, and then co‐cultured with or without Tregs at a ratio of 4:1 on a 96 well‐plate. The wells were also supplemented with IL‐2 (5 ng/mL, BD), anti‐human CD3 antibodies (1 ng/mL; eBioscience), and anti‐human CD28 antibodies (1 ng/mL; eBioscience). Sample data was acquired for flow cytometry analysis 6 days after cells incubation with 1 µmol/L indirubin or 1‰ DMSO. The data was analyzed using Flow Jo software.

Zhao Y., Han P., Liu L., Wang X., Xu P., Wang H., Yu T., Sun Y., Li L., Sun T., Liu X., Zhou H., Qiu J., Wang L., Peng J., Xu S, & Hou M. (2019). Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia. Journal of Cellular and Molecular Medicine, 23(3), 1885-1898.

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Evaluating Treg and Teff Cell Suppression with Decitabine

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CD4⁺CD25^– T cells (Teff cells) and CD4⁺CD25⁺CD127^– T cells (Treg cells) were isolated from PBMCs by using a CD4⁺CD25⁺CD127^– Regulatory T Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. CD4⁺CD25⁻ T cells were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, 2.5 μM; MilliporeSigma) and seeded at 2 × 10⁵ cells per well on a 96-well plate with or without Treg cells (Teff:Treg, 4:1). Cells were also stimulated with recombinant human IL-2 (5 ng/mL; R&D Systems), anti-human CD3 antibodies (1 ng/mL; eBioscience), and anti-human CD28 antibodies (1 ng/mL; eBioscience). In vitro, cells were harvested for proliferation analysis after incubation with 100 nM decitabine or phosphate-buffered saline for 6 days. We also sorted and cocultured Treg and Teff cells from patients before and after decitabine treatment to evaluate the suppressive function changes in vivo. Data were analyzed by using FlowJo software (FlowJo LLC, Ashland, OR).

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Isolation and Indirubin Treatment of PBMCs

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Peripheral blood mononuclear cells (PBMCs) from the patients and healthy controls were isolated from heparinized venous peripheral blood using Ficoll–Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ, USA). Isolation of circulating CD4⁺ T cells was performed using anti‐CD4‐coated magnetic beads and MS column (Miltenyi Biotec, Bergisch Gladbach, Germany) separation. The purity of the isolated cells was found to be >90% by flow cytometry.
PBMCs or CD4⁺ T cells were resuspended in RPMI‐1640 medium (Life Technologies, Paisley, UK) supplemented with 10% heat‐inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (Solarbio, Beijing, China), and recombinant human IL‐2 (5 ng/mL, R&D Systems, Minneapolis, MN, USA), anti‐human CD3 antibodies (1 ng/mL; eBioscience, San Diego, CA, USA), and anti‐human CD28 antibodies (1 ng/mL; eBioscience). The cells were then seeded on 24‐well plates.
Indirubin was dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich) to generate a stock solution of 5 mg/mL. Cultures of PBMCs or CD4⁺ T cells were treated with indirubin at a concentration of 0.01, 0.1, 0.2, 1, 2, and 10 µM, whereas 1‰ DMSO was used as vehicle controls. After 72 hours of incubation with indirubin or DMSO, PBMCs or CD4⁺ T cells were collected for flow cytometry, RNA extraction, and western blotting.

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Isolation and Activation of PBMCs

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Ficoll–Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ, USA) was used to isolated peripheral blood mononuclear cells (PBMCs) of SCLC patients and healthy controls. The isolated cells were resuscitated in RPMI-1640 medium (Life Technologies, Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (Solarbio, Beijing, China). Co-cultured with recombinant human IL-2 (10 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-human CD3 antibody (1 ng/mL; eBioscience, San Diego, CA, USA) and anti-human CD28 antibody (1 ng/mL; eBioscience).

An N., Wang H., Jia W., Jing W., Liu C., Zhu H, & Yu J. (2019). The prognostic role of circulating CD8+ T cell proliferation in patients with untreated extensive stage small cell lung cancer. Journal of Translational Medicine, 17, 402.

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Expansion of Activated T Cells

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Human peripheral blood mononuclear cells were isolated by density gradient centrifugation using a Ficoll-Paque (GE Healthcare). Cells were stimulated with 500 IU/ml of IL2 (Novartis, Basel, Switzerland), 100 ng/ml of anti-human CD3 antibody (eBioscience, San Diego, CA, USA), and 100 ng/ml of anti-human CD28 antibody (eBioscience) for 48 h. The retrovirus encoding YYB-103 was incubated for 2 h on six-well plates coated with RetroNectin (Clontech, Mountain View, CA, USA), and stimulated cells were added to the retrovirus-coated well and incubated for 24 h for transduction. Cells were maintained at 37°C and 6% CO₂ in cultured media for 9 days. Activated untransduced (UnTd) T cells from the same donors were used as controls in all experiments.

Kim K., Gwak H.S., Han N., Hong E.K., Choi B.K., Lee S., Choi S., Park J.H., Seok J.H., Jeon Y., Cho H., Lee S.J., Lee Y., Nam K.T, & Song S.W. (2021). Chimeric Antigen Receptor T Cells With Modified Interleukin-13 Preferentially Recognize IL13Rα2 and Suppress Malignant Glioma: A Preclinical Study. Frontiers in Immunology, 12, 715000.

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Isolation and Culture of NSCLC Cells

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NSCLC cells were isolated from surgical tissues with Clonogenic Tumor Cell Isolation Kit (Cell Biolabs) [20 (link)]. Digestion of NSCLC tissues was performed as previously described [41 (link)], and tumor infiltrating lymphocytes (TILs) were isolated with CD45 MicroBeads, human (Miltenyi Biotec). Depletion of macrophages from TILs was performed by using CD14 Microbeads, Human (Miltenyi Biotec). Cells were cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS (Gibco) supplemented with 2mM glutamine, 100IU/ml penicillin and 100 mg/ml streptomycin sulfate at 37°C under 5% CO2. Kits were used according to the manual's instructions.
Recombinant human IL-33 protein, human M-CSF protein, human IL-10 protein, human IL-33 neutralizing antibody and human ST2 neutralizing antibody were from R&D Systems. Anti-human CD3 antibody and anti-human CD28 antibody were from eBioscience. All reagents were used according to manufacturer's instructions.

Wang K., Shan S., Yang Z., Gu X., Wang Y., Wang C, & Ren T. (2017). IL-33 blockade suppresses tumor growth of human lung cancer through direct and indirect pathways in a preclinical model. Oncotarget, 8(40), 68571-68582.

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T Cell Activation Assay

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96-well plate was coated with 5μg/ml of anti-human CD3 antibody (eBioscience, Cat. No 16-0037) for 2 hours at 37^oC. Successively, cells were prepared and added to the plate (150.000 cells/well). Immediately after, 2μg/ml of anti-human CD28 antibody (eBioscience, Cat. No 16-0289) was added to each well and plate was incubated at 37^oC for 4 days.

Tanaskovic O., Verga Falzacappa M.V, & Pelicci P.G. (2019). Human cord blood (hCB)-CD34+ humanized mice fail to reject human acute myeloid leukemia cells. PLoS ONE, 14(9), e0217345.

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Murine Model of Cytokine Regulation

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OVA powder was purchased from Sigma-Aldrich (Grade V; St Louis, MO, USA). Adjuvant aluminum hydroxide (Al(OH)3(Alum)) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human IL-27 antibody (rhIL-27), recombinant mouse IL27 antibody (rmIL-27), recombinant human IL-4 antibody (rhIL-4), recombinant mouse IL-4 antibody (rmIL-4), recombinant human IL-2 antibody (rhIL-2), recombinant mouse IL-2 antibody, anti-human INF-γ antibody (rmIL-2), anti-mouse INF-γ antibody, anti-human IL-4 antibody and anti-mouse IL-4 antibody were obtained from R&D Systems (Minneapolis, MN, USA). Anti-human CD3 antibody, anti-mouse CD3 antibody, anti-human CD28 antibody and anti-mouse CD28 antibody were obtained from eBioscience (San Diego, CA, USA). The following antibodies were also used: anti-STAT1 antibody (SC592; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Py-STAT1 antibody (catalog no. 9171; Cell Signaling, Boston, MA, USA), anti-GADD45γ antibody (ab196774,Abcam,Cambridge,UK), anti-p38 MAPK antibody (CST9212, Cell Signaling, Boston, MA, USA), and anti-phospho-p38 MAPK antibody (CST9215, Cell Signaling, Boston, MA, USA). The concentrations of IL-4, IL-5, and IL-13 were determined using ELISA kits (eBioscience).

Su X., Pan J., Bai F., Yuan H., Dong N., Li D., Wang X, & Chen Z. (2016). IL-27 attenuates airway inflammation in a mouse asthma model via the STAT1 and GADD45γ/p38 MAPK pathways. Journal of Translational Medicine, 14, 283.

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