Soluble anti cd28

Manufactured by BioXCell

Sourced in United States

Soluble anti-CD28 is a laboratory reagent used in immunological research. It is an antibody that binds to the CD28 receptor on T cells, which plays a role in T cell activation and regulation. The core function of soluble anti-CD28 is to provide a stimulus for T cell activation and proliferation in experimental settings.

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Lab products found in correlation

Anti cd3, by BioXCell (8 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Facsaria 3 cell sorter, by BD (3 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Rmil 7, by BioLegend (2 mentions) Control rigg1, by BioLegend (2 mentions) Rmil 2, by BioLegend (2 mentions) Anti il 13, by Thermo Fisher Scientific (2 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions) Anti il 4, by BioLegend (2 mentions) Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (2 mentions) Anti cd3ε, by BioLegend (2 mentions) Rmil 25, by Johnson & Johnson (2 mentions) Anti ifn γ, by BioXCell (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Anti cd3, by BioLegend (2 mentions)

Spelling variants of this product

Anti-CD28 (62 citations) Soluble anti-CD28 antibody (5 citations) Soluble anti-CD28 (clone 37.51, (3 citations) Soluble anti-mouse CD28 (clone 37.51, (2 citations) Soluble anti-CD28 (37.51; (2 citations)

19 protocols using soluble anti cd28

Naïve T Cell Activation and Treg Differentiation

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Mice were sacrificed, their spleens were removed and gently dissociated into single-cell suspensions. Naïve T cells were isolated using the MojoSort Mouse CD4 Naïve T Cell Isolation Kit (BioLegend). T cells were cultured in U-bottom 96-well plates with RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM l-glutamine, 1% antibiotic-antimycotic (Gibco), 10 mM HEPES (Gibco), 1× nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and 55 μM β-mercaptoethanol (Gibco). Naïve T cells were activated with plate-bound anti-CD3 (3 μg/ml; Bio X Cell) and soluble anti-CD28 (2 μg/ml; Bio X Cell) antibodies. T_reg cell differentiation was achieved by the indicated concentration of recombinant human TGF-β (R&D). The Akt inhibitor MK-2206 and afuresertib were purchased from MedChemExpress (MCE). Cells were cultured for 72 h and detected by flow cytometry.

Cai W., Zhang J., Zhou H., Li X., Lou F., Sun Y., Xu Z., Bai J., Yin Q., Wang Z., Sun L., Cai X., Tang S., Wu Y., Fan L., Wang H., Wang H, & Li Q. (2021). Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity. Genes & Diseases, 9(2), 562-575.

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CD8+ T Cell Adoptive Transfer Protocol

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CD8⁺ T cells were purified from the spleens of naive CD45.1⁺ OT-I mice using EasySep Mouse CD8⁺ T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Single T cell preparations were >95% pure as determined by flow cytometry. Effector T cells were generated by activating naive T cells for32 h with plate-bound anti-CD3 (10 μg/ml;145–2C11; Bio X Cell), soluble anti-CD28 (5 μg/ml; 37.51; Bio X Cell), and rIL-2 (25 U/ml; National Cancer Institute). Cells were transduced (8 × 10⁶ cells/ml) with the different viral supernatants with polybrene (8 μg/ml), then cultured in RPMI 1640 with 10% FBS in the presence of IL-2 (25 U/ml) for 48 h. Effector CD8⁺ T cells (5 × 10⁴/mouse) were injected retro-orbitally into LM-ActA⁻OVA–infected recipients, and the splenocytes were collected on day 7 and day 28 postinfection.

Hwang J.Y., Holland J.E., Valenteros K.B., Sun Y., Usherwood Y.K., Verissimo A.F., McLellan J.S., Grigoryan G, & Usherwood E.J. (2019). Dissociating STAT4 and STAT5 Signaling Inhibitory Functions of SOCS3: Effects on CD8 T Cell Responses. ImmunoHorizons, 3(11), 547-558.

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Primary B Cell Activation Protocols

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For LPS/IL4 cultures, primary B cells were purified by immunomagnetic depletion using anti-CD43 beads (Miltenyi Biotec) and cultured at a final concentration of 1.2 × 10⁶ cells per ml in complete RPMI supplemented with 10% FBS, 50 μM of 2-βMercaptoethanol (Gibco), 20 mM Hepes (Gibco), 10 ng ml⁻¹ of IL4 (PeproTech) and 25 μg ml⁻¹ lipopolysaccharide (LPS, Sigma-Aldrich). In the T–B-cell co-culture, CD43⁺ cells were added at a ratio 1:1 to CD43⁻ B cells in the presence of plastic-bound anti-CD3 (Tonbo, 5 μg ml⁻¹) and soluble anti-CD28 (BioXcell, 2 μg ml⁻¹) for T-cell stimulation. When indicated, cells were treated with anti-mouse CD40 (BD Pharmigen, 1 μg ml⁻¹).

Pérez-García A., Marina-Zárate E., Álvarez-Prado Á.F., Ligos J.M., Galjart N, & Ramiro A.R. (2017). CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation. Nature Communications, 8, 16067.

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Isolation and Stimulation of Mouse Splenic T Cells

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Mouse spleen CD3⁺ T cells were isolates by using MojoSort Mouse CD3 T Cell Isolation Kit (Biolegend, #480031) resulting in >95% purity. Isolated spleen CD3⁺ T cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, #C34554) for 10 min at room temperature (RT) and then blocked with 3 mL FBS for 10 min at 37°C. CFSE-labeled T cells were resuspended in culture medium (RPMI 1640 supplemented with 10% PBS, 1% L-Glutamine, 1% penicillin-streptomycin and 10% PBS, 1% L-Glutamine, 1% penicillin-streptomycin and 0.055mM 2-mercaptoethanol) and stimulated with 2.5 μg/mL coated anti-CD3 antibody (Bio X Cell, #BE0001-1) and 2.5 μg/mL soluble anti-CD28 (Bio X Cell, #BE0015-1) antibody in U-bottom 96-well plates for 2 days. For coculture studies, CFSE-labeled purified T cells (1 × 10⁶ cells/mL) were mixed with 5-day PBS or H-LPS treated WT or TRAM KO monocytes at a ratio of 1:1 in the presence of coated anti-CD3 and soluble anti-CD28 antibody for 2 days in a U-bottom 96-well plates (200 μL/well).

Wang J., Wu Y., Lin R., Zhang Y, & Li L. (2023). TRAM deletion attenuates monocyte exhaustion and alleviates sepsis severity. Frontiers in Immunology, 14, 1297329.

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Intracellular TNFα Staining in Activated Splenocytes

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Total splenocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 100U/ml penicillin-streptomycin (Life Technologies) and 2mM L-glutamine (Life Technologies), and stimulated as follows. For TNFα intracellular staining, 5 × 10⁶ thymocytes or splenocytes were stimulated with or without plate-coated anti-CD3ε (10 μg/ml, 145-2C11, Bio-X-Cell) plus soluble anti-CD28 (1μg/ml, 37.51, Bio-X-Cell) in the presence of Protein Transport Inhibitor Cocktail (eBioscience) for 17 hours at 37°C in 5% CO₂. After the incubation, cells were harvested and washed once with 1X PBS, and stained with fixable viability dye before surface staining. Following the surface stain, cells were fixed and permeabilized with IC Fixation Buffer (eBioscience) and then stained for intracellular TNFα (MP6-XT22, eBioscience).

Hsu F.C., Belmonte P., Constans M., Chen M.W., McWilliams D.C., Hiebert S.W, & Shapiro V.S. (2015). HDAC3 is required for T cell maturation. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1578-1590.

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CD8+ T Cell Adoptive Transfer Protocol

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Isolation and Stimulation of Naïve CD4+ T Cells

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CD4⁺ T cells from the spleen and peripheral LNs were positively selected with CD4 microbeads (L3T4; Miltenyi Biotec), and naive CD4⁺ T cells were further sorted as CD4⁺CD25⁻CD62L^highCD44^low cells with the FACSAria III cell sorter (Becton, Dickinson and Company (BD) Biosciences) and stimulated with plate-coated anti-CD3 (1 μg/mL, 145-2C11: BioXCell) and soluble anti-CD28 (1 μg/mL, 37.51; BioXCell) for 3 d in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich). To measure TCR-mediate protein phosphorylation, flow-sorted naïve CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 (2 μg/mL) before being analyzed at indicated time points. In some experiments, cells were treated with either DMSO vehicle or 1 µM GW3965 (Tocris), 1 µM BIO (Sigma-Aldrich), or mevalonic acid (Sigma-Aldrich).

Kim J., Lee H., Lee J.E., Choi G., Chung H., Kim D., Park M.J., Gye Y.S., Shin K.S., Kang C.Y., Kwok S.K, & Chung Y. (2023). Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1. Proceedings of the National Academy of Sciences of the United States of America, 120(9), e2213793120.

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Isolation and polarization of T cell subsets

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Naïve CD4 T cells were isolated from mouse spleens using the CD4⁺CD62L⁺ T cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec). Cells were cultured in R10 medium on anti-CD3 (2 µg/ml; BioXCell) coated cell culture plates with soluble anti-CD28 (2 µg/ml; BioXcell). Cells were cultured under Treg polarizing conditions including hTGF-β1 (2 ng/ml), hIL-2 (50 U/ml), anti-IFN-γ (XMG; 10 mg/ml) and anti-IL-4 (11B11; 10mg/ml). Th9 cells were cultured with hTGF- β1 (2 ng/ml), IL-4 (20 ng/ml), hIL-2 (50 U/ml) and anti-IFN-γ (10 mg/ml). Th0 cells were cultured with hIL-2 (50 U/ml), anti-IFN-γ (XMG; 10 mg/ml) and anti-IL-4 (11B11; 10mg/ml). On day 3, cells were expanded into fresh media containing the original concentrations of cytokines in the absence of co-stimulatory signals for additional 2 days. On day 5, mature T cell subsets were harvested for further analysis.

Heim L., Yang Z., Tausche P., Hohenberger K., Chiriac M.T., Koelle J., Geppert C.I., Kachler K., Miksch S., Graser A., Friedrich J., Kharwadkar R., Rieker R.J., Trufa D.I., Sirbu H., Neurath M.F., Kaplan M.H, & Finotto S. (2022). IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer. Frontiers in Immunology, 13, 859738.

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Purification and Activation of CD8 T Cells

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CD8 cells were purified from spleen and lymph nodes (LN) by negative selection
as previously described (Farley et al., 2006 (link)) and
by positive selection using the MACS Cell Separation System (Miltenyi) as recommended by
the manufacturer. The purity of isolated cells was confirmed by flow cytometry. Cells were
activated in vitro using plate bound anti–CD3 (5 μg/mL)
and soluble anti–CD28 (1 μg/mL) antibodies (BioXcell). For analysis of
cytokine secretion during resting, cells were activated for 2 days, washed, and incubated
at equal numbers in medium alone without additional stimuli for 4 – 12h. Culture
supernatants of activated and rested cells were analyzed for IFNγ, IL-2, or GM-CSF
by ELISA. Cells were also incubated with or without oligomycin (20 μM, Sigma), or
cycloheximide (5 μg/ml, Sigma). For cell survival analysis, activated cells were
washed and incubated at equal numbers in medium without further stimulation or the
presence of exogenous cytokines. In some cases, the culture medium was supplemented with
anti-IL-2 Ab or recombinant IL-2 after 24 h. Live cells were determined by Trypan blue
exclusion at 24, 48, and 72 h after replating.

Champagne D.P., Hatle K.M., Fortner K.A., D’Alessandro A., Thornton T.M., Yang R., Torralba D., Tomás-Cortázar J., Jun Y.W., Ahn K.H., Hansen K.C., Haynes L., Anguita J, & Rincon M. (2016). Fine-tuning of CD8 T cell mitochondrial respiration by MCJ/DnaJC15 dictates protection to influenza virus. Immunity, 44(6), 1299-1311.

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ILC2-Mediated Modulation of Tumor M-MDSCs

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Intestinal ILC2s were sorted and cultured in complete RPMI (RPMI-1640 + 10% FCS + penicillin/streptomycin + 2-mercaptoethanol) with 10 ng/mL rm-IL-2 (Biolegend, 575406), 10 ng/mL rm-IL-7 (Biolegend, 577806), and 20 ng/mL rm-IL-25 (Janssen) for 3 days at 37°C, and supernatant collected (ILC2-SNT). ILC2-SNT were incubated with anti-IL-4 (Biolegend, 504122) and anti-IL-13 (eBioscience, 16-7135-85) neutralizing antibodies (αIL-4/13Ab-ILC2-SNT), or control rIgG1 (Biolegend, 400432 and eBioscience, 16-4301-85) antibodies (conAb-ILC2-SNT) for 1 hour on ice. Tumor M-MDSCs were sorted and incubated with αIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT for 2 hours at 37°C. Subsequently, sorted splenic CD8⁺ T cells (Live CD45⁺ TCRγδ^-CD4^-CD8⁺) stained with the CellTrace Violet Cell Proliferation Kit (ThermoFisher, C34557), were added to the ILC2-SNT-M-MDSC culture, and stimulated for 3 days with plate-coated anti-CD3ε (Biolegend, 100360) and 2 μg/mL soluble anti-CD28 (Bio X Cell, BE0015-1) at 37°C. Cell Stimulation Cocktail (eBioscience, 00-4975-93) was added for the last 4 hours of culture, before antibody staining. For Arginase 1 expression analysis in ILC2-SNT-treated M-MDSCs, tumor or splenic M-MDSCs were sorted and cultured with αIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT for 2 days, and stained with antibodies for flow cytometry.

Jou E., Rodriguez-Rodriguez N., Ferreira A.C., Jolin H.E., Clark P.A., Sawmynaden K., Ko M., Murphy J.E., Mannion J., Ward C., Matthews D.J., Buczacki S.J, & McKenzie A.N. (2022). An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc-mutation-driven intestinal tumorigenesis. Science immunology, 7(72), eabn0175.

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