Soluble anti cd28 antibody

Manufactured by BioLegend

Sourced in United States

The Soluble anti-CD28 antibody is a laboratory reagent designed to activate T cells by engaging the CD28 receptor. CD28 is a co-stimulatory receptor expressed on the surface of T cells that, when bound, provides a secondary signal required for full T cell activation. This soluble antibody can be used in cell culture experiments to stimulate T cell proliferation and cytokine production.

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Lab products found in correlation

Anti cd3 antibody clone okt3, by BioLegend (3 mentions) Interleukin 2 (il 2), by R&D Systems (3 mentions) Anti cd3 antibody, by BioLegend (2 mentions) 96 u bottom well plates, by BD (2 mentions) Clone md 1, by BioLegend (2 mentions) Tgf β, by R&D Systems (2 mentions) Bexarotene, by Abcam (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Pre coated anti cd3 antibody, by BioLegend (2 mentions) Il 12, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Interleukin 2 (il 2), by BioLegend (2 mentions) Immunomagnetic separation beads, by Miltenyi Biotec (1 mentions) Cell stimulation cocktail, by Cytek Biosciences (1 mentions) Af 100 21c, by Thermo Fisher Scientific (1 mentions) Sodium butyrate, by Merck Group (1 mentions) Tgf β1, by R&D Systems (1 mentions) Il 1β, by R&D Systems (1 mentions) Interleukin 6 (il 6), by R&D Systems (1 mentions)

Close spelling variants of this product

Anti-CD28 (244 citations) Anti-CD28 antibody (54 citations) Soluble anti-CD28 (52 citations) CD28 antibody (12 citations) Soluble anti-CD28 antibody (clone CD28.2, (2 citations)

16 protocols using soluble anti cd28 antibody

Th17 Cell Differentiation from Naive CD4+ T Cells

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Naïve CD4 cells were isolated from C57BL/6J mouse spleens using immunomagnetic separation beads (Miltenyi Biotec, 130-106-643) according to the manufacturer's instructions and seeded into 96-well plates (5 × 10⁵/well) precoated with anti-CD3 antibody (5 μg/ml) (BioLegend, 100309) with the addition of soluble anti-CD28 antibody (2 μg/ml) (BioLegend, 102102). MSCs, sh-NC-MSCs, sh-Chi3l1-MSCs, or sh-Chi3l1-MSCs plus Stattic (20 μM, Selleck, S7024) were seeded into 96-well plates (5 × 10³/well) 6 h before CD4 cell seeding. Th17 differentiation medium contained TGF-β (1.0 ng/ml) (PeproTech, AF-100-21C), IL-6 (30 ng/ml) (PeproTech, 216-16), IL-1β (20 ng/ml) (PeproTech, 211-11B), IL-23 (20 ng/ml) (BioLegend, 589002), anti-IL-4 (10 μg/ml) (BioLegend, 504102), and anti-IFN-γ (10 μg/ml) (BioLegend, 505833).
Th17 cells were differentiated for 72 h and restimulated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975) for 6 h before further analysis for intracellular cytokines by flow cytometry.

Liu W., Yuan F., Bai H., Liu Y., Li X., Wang Y, & Zhang Y. (2022). hUC-MSCs Attenuate Acute Graft-Versus-Host Disease through Chi3l1 Repression of Th17 Differentiation. Stem Cells International, 2022, 1052166.

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Optimizing Induced Regulatory T Cell Generation

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After MACS isolation, naive T cells were rested for 3 to 8 hours and then plated under iTreg differentiation conditions at 1.1 to 1.3x10⁵ cells/well in 96U well plates. Cells were stimulated with 5 μg/ml plate-bound anti-CD3 antibody (clone OKT3; Biolegend, LEAF grade), 1 μg/ml soluble anti-CD28 antibody (Biolegend, LEAF grade) and 100 IU/ml IL-2 (carrier-free; R&D Systems). Cells stimulated with only these reagents served as “mock” control. For Treg-inducing conditions, TGF-β1 (5 ng/ml carrier-free; R&D Systems), ATRA (10 nM; Sigma-Aldrich), Rapa (100 ng/ml; Calbiochem EMD Millipore), sodium butyrate (100 μM; Sigma-Aldrich), or STAT3 inhibitor S3I-201 (50 μM; Sigma-Aldrich) were added additionally. The DMSO control (for ATRA, Rapa, STAT3i) had no effect on Foxp3 expression. Cells were incubated for 6 days unless otherwise stated.

Schmidt A., Eriksson M., Shang M.M., Weyd H, & Tegnér J. (2016). Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate. PLoS ONE, 11(2), e0148474.

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Th17 Cell Differentiation Protocol

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After either MACS or FACS isolation, naive cells were plated under Th17 differentiation conditions at 5x10⁵ cells/well in 96 U-bottom well plates (Falcon). For cell stimulation, plates were coated at least 5 hours prior to use with 3 ug/ml plate-bound anti-CD3 antibody (clone OKT3; BioLegend, LEAF grade), 1 ug/ml soluble anti-CD28 antibody (BioLegend, LEAF grade) and 2 ng/ml IL-2 (carrier-free; Tocris R&D Systems). Cells treated with only these functioned as the mock control. For Th17 induction, TGF- β (30 ng/ml; carrier-free; Tocris R&D Systems), IL-6 (30 ng/ml; carrier-free; Tocris R&D Systems), IL-1 β (10 ng/ml; carrier-free; Tocris R&D Systems), IL-23 (50 ng/ml; carrier-free; Tocris R&D Systems), anti-IFN γ antibody (2 ug/ml; clone MD-1, Biolegend), anti-IL-4 antibody (2 ug/ml; clone MD-1, Biolegend), bexarotene (1 µM, Abcam), or atRA (100 nM; Sigma-Aldrich) were added additionally. The DMSO control (for bexarotene, atRA and AGN190) had no effect on IL-17A cytokine expression. Cells were incubated for 7 days unless otherwise stated.

Gaunt C.M., Rainbow D.B., Mackenzie R.J., Jarvis L.B., Mousa H.S., Cunniffe N., Georgieva Z., Brown J.W., Coles A.J, & Jones J.L. (2021). The MS Remyelinating Drug Bexarotene (an RXR Agonist) Promotes Induction of Human Tregs and Suppresses Th17 Differentiation In Vitro. Frontiers in Immunology, 12, 712241.

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Expansion of Tumor-Infiltrating Lymphocytes

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Enriched TIL fractions (~50% T cells) were induced to proliferate in a 24‐well plate (1 × 10⁶/well) that had been immobilized with anti‐CD3 antibody (1 μg/mL) in the presence of soluble anti‐CD28 antibody (0.5 μg/mL, both rat anti-mouse, BioLegend) in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, 2 mM l-glutamine, 50 μM β-mercaptoethanol. After 24 h (day 2), the culture was supplemented with 50 IU/ml recombinant IL-2 (rIL2, Hoffmann La Roche, kindly provided by the National Cancer Institute Biological Resources Branch Preclinical Repository, Rockville, MD). To accommodate the rapid T cell proliferation, cells were replenished with a new medium containing IL-2 every three days and the cell density was maintained at 1 × 10⁶ cells/ml. Cells were harvested on day 10 since T cells often had proliferated more than eight generations and had expanded over 100-fold. These expanded T cells, of which 70–80% were Tc, were termed TIL-MC38 or TIL-KPC dependent on the tumor from which the initial TILs were isolated, and these expanded TILs were further tested for tumor cytotoxicity in vitro and in vivo.

Bian Z., Shi L., Kidder K., Zen K., Garnett-Benson C, & Liu Y. (2021). Intratumoral SIRPα-deficient macrophages activate tumor antigen-specific cytotoxic T cells under radiotherapy. Nature Communications, 12, 3229.

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Naïve CD4+ T Cell Polarization

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Naive CD4⁺ T cells from the spleens of 6-8 weeks old male GREAT mice were prepared using magnetic bead cell sorting (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of CD4⁺CD44^lowCD62L^high T cell subset was validated by flow cytometry. These naïve CD4⁺ T cells were stimulated with 5 μg/ml pre-coated anti-CD3ϵ antibody (Biolegend) and 1 μg/ml soluble anti-CD28 antibody (Biolegend) in culture medium containing 10 ng/ml IL-12 (Peprotech, Cranbury, NJ, USA), 10 ng/ml IL-2 (Peprotech) and 10 μg/ml anti–IL-4 antibody (BD Biosciences) for 3 days. The culture medium was RPMI 1640 medium (plus 50 μM β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). 1 × 10⁵ naive CD4⁺ T cells were cultured in 96-well plates with 100 μl culture medium per well.

Zeng Q., Song J., Wang D., Sun X., Xiao Y., Zhang H., Xiao Y., Zhou Z, & Deng T. (2022). Identification of Sorafenib as a Treatment for Type 1 Diabetes. Frontiers in Immunology, 13, 740805.

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Differentiation of Induced Regulatory T Cells

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To investigate the differentiation of iTreg, CD4⁺ naïve T cells were purified using a commercially available isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Germany) and stimulated with plate-coated anti-CD3 antibody (1 mg/ml, Biolegend, USA) and soluble anti-CD28 antibody (1 mg/ml, Biolegend, USA) in X-VIVO15 medium (Lonza, Switzerland). Three days after plating, TGF-b (1 ng/ml, Biolegend, USA) and interleukin 2 (IL-2, 50 U/ml, Biolegend, USA) were added in the culture, and the cells were continued to cultivate for 2 days. According to different experimental purposes, reagents were added together with the polarization cytokines, including methyl-b-cyclodextrin-cholesterol (cholesterol, 10–20 mg/ml, Sigma, USA), dimethyloxalylglycine (DMOG, 100 mM, MCE, USA), PX-478 (10 mM, MCE, USA), MT (10 mM, Sigma, USA) and IL-1b (100 ng/ml, Biolegend, USA).

Zhang H., Xia N., Tang T., Nie S., Zha L., Zhang M., Lv B., Lu Y., Jiao J., Li J, & Cheng X. (2023). Cholesterol suppresses human iTreg differentiation and nTreg function through mitochondria-related mechanisms. Journal of Translational Medicine, 21, 224.

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Enhancing CD8 T Cell Activation

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Human CD8 T cells were seeded in anti-CD3 (1 μg/ml, BD Biosciences) pre-coated 96-well plates and cultured with conditions where indicated with the following reagents: 1) 1 μg/ml soluble anti-CD28 antibody (Biolegend); 2) 100 ng/ml of soluble recombinant MICB (Sino Biologicas); 3) 100 ng/ml of B10G5. IFNγ production was assayed by intracellular staining after 24 h of culture (BD IFNγ staining Kits).
For assessing antigen-specific CD8 T cell response, human tyrosinase-specific HLA-A₂-restricted TIL13831 was co-cultured o/n with the HLA-A2⁺ T2-A2 cells (Generous gifts of Dr. Rubinstein at the Medical university of South Carolina) under indicated condition before functional assay. The tyrosinase peptide_369–377 was purchased from AnaSpec (Fremont, CA). After overnight culture, activation of TIL13831 was assessed by intracellular staining for IFNγ, TNFα, and CD107a (degranulation).

Zhang J., Larrocha P.S., Zhang B., Wainwright D., Dhar P, & Wu J.D. (2019). Antibody targeting tumor-derived soluble NKG2D ligand sMIC provides dual co-stimulation of CD8 T cells and enables sMIC+ tumors respond to PD1/PD-L1 blockade therapy. Journal for Immunotherapy of Cancer, 7, 223.

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Murine CD4+ T-cell Activation and Stimulation

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Unless otherwise stated, cells were cultured in RPMI 1640 medium (Gibco, New York, USA) containing 10% foetal bovine serum (FBS; HyClone, Los Angeles, USA), 1% 1 M HEPES, 2 mm glutamine (Sigma‐Aldrich, Milwaukee, USA), 100 U mL⁻¹ penicillin (Sigma‐Aldrich), 0.1 mg mL⁻¹ streptomycin (Sigma‐Aldrich), 1% 100 × sodium pyruvate and 2 μm 2‐mercaptoethanol (Invitrogen, Carlsbad, USA) in a humidified atmosphere (37°C and 5% CO₂). Activated CD4⁺ T cells were purified magnetically from splenocytes isolated from wild‐type C57BL/6 mice aged 6–8 weeks (purity > 90%) using a CD4⁺ T‐cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured at 1 × 10⁶ per well in the presence of plate‐bound anti‐CD3 antibody (2 µg mL⁻¹, Biolegend, California, USA) and soluble anti‐CD28 antibody (2 µg mL⁻¹, Biolegend) for activation.⁵⁶ Inactive CD4⁺ T cells were cultured in the absence of anti‐CD3 and anti‐CD28 antibodies. Different concentrations of Pg or LGG bacterial extracts were added to the culture medium to stimulate cells for subsequent detection via flow cytometry, RT‐qPCR or enzyme‐linked immunosorbent assay (ELISA).

Jia L., Wu R., Han N., Fu J., Luo Z., Guo L., Su Y., Du J, & Liu Y. (2020). Porphyromonas gingivalis and Lactobacillus rhamnosus GG regulate the Th17/Treg balance in colitis via TLR4 and TLR2. Clinical & Translational Immunology, 9(11), e1213.

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In Vitro Differentiation of Naive CD4+ T Cells

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Healthy volunteers were recruited from the medical students at the Second Xiangya Hospital and written informed consent was obtained. Human study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy volunteers were separated by Histopaque (Sigma-Aldrich, St. Louis, MO). Naive CD4⁺ T cells from PBMC were isolated by negative selection using Miltenyi beads according to the manufacturer’s instructions. The purity of CD4⁺CD45RA⁺CD45RO^- T cell subset was validated by flow cytometry. The purified naïve CD4⁺ T cells were stimulated with 5 μg/ml pre-coated anti-CD3ϵ antibody (Biolegend) and 2 μg/ml soluble anti-CD28 antibody (Biolegend) in culture medium containing 10 ng/ml IL-12 (Peprotech, Cranbury, NJ, USA), 10 ng/ml IL-2 (Peprotech) and 10 μg/ml anti–IL-4 antibody (eBioscience) for 5 days. The culture medium was RPMI 1640 medium supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). 1 × 10⁵ naive CD4⁺ T cells were cultured in 96-well plates with 100 μl culture medium per well. The culture medium was refreshed on day 3.

Zeng Q., Song J., Wang D., Sun X., Xiao Y., Zhang H., Xiao Y., Zhou Z, & Deng T. (2022). Identification of Sorafenib as a Treatment for Type 1 Diabetes. Frontiers in Immunology, 13, 740805.

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Polarization of Murine CD4+ T Cells

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Naïve CD4⁺ T cells were isolated from spleen cell suspensions using mouse naïve CD4⁺ T cell isolation kits from Miltinyi Biotec per manufacturer’s instruction. All CD4s subjected to subset polarization were resuspended in complete media into 6 well plates at a seeding density of 1x10⁶/ml and stimulated with immobilized anti-CD3 antibody (BioLegend; clone 145-2C11) plus soluble anti-CD28 antibody (BioLegend; clone 37.51) followed by the addition of reagents designed to skew the CD4 cells towards a Th1, Th17, or Treg phenotype according to manufacturer’s specifications from kits purchased from BioLegend. Briefly, for Th1 polarization, anti-mouse IL-4, clone 11B11 (10 μg/ml), recombinant mouse IL-2 (5 ng/ml) and recombinant mouse IL-12 (10 ng/ml) were added to the cultures. To promote differentiation into Th17 subsets, recombinant mouse IL-6 (50 ng/ml), recombinant human TGF-β (1 ng/ml), recombinant mouse IL-23 (5 ng/ml), anti-mouse IL-4 (10 μg/ml), and anti-mouse IFN-γ (10 μg/ml) were added to the cultures. For regulatory T cell (T reg) polarization, the cultures were treated with recombinant mouse IL-2 (5 ng/ml) and recombinant human TGF-β1 (5 ng/ml). On day 4, cells were harvested, and RNA were isolated for qRT-PCR assessment for subset-specific gene expression.

Blossom S.J., Cabanlong C.V, & Vyas K.K. (2022). Developmental trichloroethylene exposure enhances predictive markers of autoimmunity in a sex-specific manner in disease-resistant female mice. Toxicology and applied pharmacology, 454, 116233.

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