Anti cd28 clone 37

Manufactured by BioLegend

Sourced in United States

Anti-CD28 (clone 37.51) is a monoclonal antibody that binds to the co-stimulatory molecule CD28 expressed on T cells. This antibody can be used to stimulate T cell activation and proliferation in vitro.

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Anti-CD28 (244 citations) Anti-CD28 (37.51, (44 citations) Clone 37.51 (33 citations) Clone CD28.2 (30 citations) Anti-CD28 (clone CD28.2, (25 citations) Anti-CD28 antibody (clone 37.51, (6 citations) Anti-mouse CD28 antibody (clone 37.51, (3 citations) Soluble anti-CD28 (clone 37.51, (3 citations) Anti-CD28 antibodies (clone 37.51 (2 citations)

25 protocols using anti cd28 clone 37

Selenium-Mediated Modulation of Th17 Cell Differentiation

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WT C57BL/6 mice or CD4^Cre Tcf7^fl/fl mice CD4⁺ naïve T cells were isolated using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and further sorted in a flow cytometer (Aria III, BD). CD4⁺CD25⁻CD44⁻CD62L⁺ naïve T cells with a purity of > 95% were used for in vitro culture experiments. Naïve CD4⁺ T cells were cultured in the anti‐CD3 (clone: 17A2, Biolegend) and anti‐CD28 (clone: 37.51, Biolegend)‐coated 96‐well plates and cultured under a Th17 cell polarisation condition: TGF‐β (1 ng mL⁻¹, PeproTech, Cranbury, NJ, USA), IL‐6 (20 ng mL⁻¹, PeproTech), anti‐IFN‐γ (10 μg mL‐⁻¹, PeproTech) and anti‐IL‐4 (10 μg mL⁻¹, PeproTech) for 72 h. Se‐Met (0, 1.25 and 5 μM) with or without NAC (2 μM, Sigma‐Aldrich) was added to the culture medium. For the CFSE‐labelled proliferation assay, naïve CD4⁺ T cells were pre‐stained with CFSE (1 μg mL⁻¹) at 37°C for 10 min and then cultured in the anti‐CD3/CD28‐coated plates for 72 h.

Qin J., Huang X., Wang N., Zhou P., Zhang H., Chen Z., Liang K., Gong D., Zeng Q., Niu P., Chen A., Yuan L., Yang Z., Su L., Shen N., Deng J, & Yu D. (2021). Supranutritional selenium suppresses ROS‐induced generation of RANKL‐expressing osteoclastogenic CD4+ T cells and ameliorates rheumatoid arthritis. Clinical & Translational Immunology, 10(9), e1338.

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Transcriptome Analysis of LCMV-Specific T Cells

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P14 cells were sorted (BD FACS Aria, BD Biosciences) from animals with either acute or chronic LCMV infection. After 4 h of in vitro restimulation in the presence of anti-CD3 (clone 145-2C11, Biolegend) and anti-CD28 (clone 37.51, Biolegend) antibodies, RNA was extracted using TRIzol (Thermofisher Scientific) according to the manufacturer’s protocol and processed for sequencing at the Functional Genomics Center Zürich (FGCZ). The extracted RNA was selected for messenger RNA (mRNA) transcripts using polyA-tail capture. The enriched mRNA was reverse transcribed and sequenced on the Illumnia HiSeq 4000 platform. Resulting transcript counts were analyzed and differential gene expression was analyzed using the DEseq2 package^{45 (link)} in R⁴⁶.

Sandu I., Cerletti D., Claassen M, & Oxenius A. (2020). Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection. Nature Communications, 11, 4454.

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Splenocyte Isolation and Activation

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Splenocytes were isolated by mechanical disruption and made into single cell suspensions in RPMI 1640 medium supplemented with 10% BCS and penicillin/streptomycin. Red blood cells were lysed using Zap-oglobin prior to counting splenocytes on a Beckman Coulter Counter following manufacturer’s instructions with a size threshold of 4.0 μm. Splenocytes were then cultured at 1 × 10⁶ cells/well in 96 well plates (for proliferation assay) or 2.5 × 10⁶ cell/well in 48 well plates (for ELISA and flow cytometry analyses). Cells were treated with 15 μg/ml LPS (S. typhosa, Sigma Aldrich, St. Louis, MO), 15 μg/ml PWM (Sigma Aldrich), or 1 μg/ml anti-CD3 (clone 145-2C11, Biolegend, San Diego, CA) plus 10 μg/ml anti-CD28 (clone 37.51, Biolegend). For anti-CD3/CD28 treatment, culture plates were coated overnight with 1 μg/ml anti-CD3 at 4 °C, washed twice with 1640 RPMI, and then seeded with cells and 10 μg/ml anti-CD28. Cells were culture at 37 °C with 5% CO2 for up to 72 hours.
Post-activation, cells were harvested by centrifugation at 500 × g for 5 minutes. Supernatants were collected for IgM ELISA, and cells were washed in Hank’s Balanced Salt Solution and stained for flow cytometry.

Li J., Bach A., Crawford R.B., Phadnis-Moghe A.S., Chen W., D’Ingillo S., Kovalova N., Suarez-Martinez J.E., Zhou J., Kaplan B.L, & Kaminski N.E. (2018). CLARITY-BPA: Effects of Chronic Bisphenol A Exposure on the Immune System: Part 2 - Characterization of Lymphoproliferative and Immune Effector Responses by Splenic Leukocytes. Toxicology, 396-397, 54-67.

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T Cell Activation Assay

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OT-I Rgs1^+/+ T cells were FACS sorted, and 1x10⁵ cells/well were distributed in a 96-U-bottom-well plate in 200μl cell culture media (RMPI 1640 + 2mM L-Ala/L-Glu, 1mM Sodium Pyruvate, 10mM HEPES, 1x MEM non-essential amino acids, 0.5mM 2-β-Mercaptothion, 10% FCS, 40 U/ml Penicillin, 40μg/ml Streptomycin (all Sigma-Aldrich, St. Louis, USA)) and stimulated with distinct cytokines and TCR-crosslinking mAbs. For TCR stimulation, plates were previously coated with anti-CD3ε and anti-CD28 mAb´s (anti-CD3ε: clone 145-2C11, anti-CD28: clone 37.51, both Biolegend, San Diego, USA) for 12h. The cells were treated with recombinant murine IL-2 (50 ng/ml, R&D Systems, Minneapolis, USA), IL-15 (50 ng/ml, Peprotech, London, United Kingdom), IL-33 (100 ng/ml, Peprotech, London, United Kingdom) and/or recombinant human TGFβ1 (50 ng/ml Peprotech, London, United Kingdom). The cells were incubated for 72h, at 37°C in 5% CO₂.

von Werdt D., Gungor B., Barreto de Albuquerque J., Gruber T., Zysset D., Kwong Chung C.K., Corrêa-Ferreira A., Berchtold R., Page N., Schenk M., Kehrl J.H., Merkler D., Imhof B.A., Stein J.V., Abe J., Turchinovich G., Finke D., Hayday A.C., Corazza N, & Mueller C. (2023). Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity. Frontiers in Immunology, 14, 1085895.

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Isolation and Activation of Mouse CD8+ T Cells

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Mouse CD8⁺ T cells were prepared as previously described.35 (link) Spleens and lymph nodes from wild-type C57BL/6 or HRH1^−/− mice were harvested and filtered through a 70 µm cell strainer to generate a single-cell suspension. CD8⁺T cells were isolated by using mouse CD8 naïve T-cell isolation kit (BioLegend) and then plated in DC/T cell medium supplemented with β-mercaptoethanol (50 µM), 20 ng/mL recombinant mouse IL-2 (PeproTech) and 10 ng/mL recombinant mouse IL-7 (PeproTech). For activated T cells, cells were cultured in 48-well plates coated with 5 µg/mL anti-CD3 (clone 145–2 C11, BioLegend) and 2.5 µg/mL anti-CD28 (clone 37.51, BioLegend) antibodies for 48 hours and then transferred to uncoated plates for at least 24 hours before use.

Zhang S., Zhao L., Guo M., Liu P., Li S., Xie W., Tian A.L., Pol J.G., Chen H., Pan H., Mao M., Li Y., Zitvogel L., Jin Y., Kepp O, & Kroemer G. (2023). Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses. Journal for Immunotherapy of Cancer, 11(7), e006785.

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Modulating T Cell Signaling Pathways

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For PP2 assay, day 6–10 blasting T cells were pre-incubated with PP2 (10 μM; 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Sigma-Aldrich) for 1 h at 37 °C and 5% CO₂. Pre-treated cells were imaged at 37 °C and 5% CO₂ in the presence of 10 μM PP2.
For CD4 and CD28 blockade, an anti-CD4 (clone Gk1.5, BioLegend) or anti-CD28 (clone 37.51, BioLegend) Fab was prepared and purified with Micro Fab Preparation Kit (Thermo Fisher Scientific). Day 6–10 T cells were pre-incubated for 1 h at 37 °C and 5% CO₂ with anti-CD4 Fab (2 μg/mL) or anti-CD28 Fab (4 μg/mL) then imaged in the presence of 2 μg/mL Fab.

Rosenberg J., Cao G., Borja-Prieto F, & Huang J. (2020). Lattice Light-Sheet Microscopy Multi-Dimensional Analyses (LaMDA) of T-Cell Receptor Dynamics Predict T-Cell Signaling States. Cell systems, 10(5), 433-444.e5.

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Murine CD4+ T Cell Activation

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All primary murine CD4⁺ T cells came from homozygous OT-II TCR transgenic mice (Taconic). The following antibodies were used in this study: biotinylated anti–human-CD25 (clone BC96), biotinylated anti-CD3ε (clone 145-2C11), biotinylated anti-CD43 (clone 1B11), and anti-CD28 (clone 37.51) from BioLegend, and anti-CD3ε (clone 145-2C11) from Bio X Cell. Biotinylated I-A^b presenting OVA(323–339) was obtained from the National Institutes of Health Tetramer Facility for pMHC studies. Ovalbumin peptide 323–339 was obtained from AnaSpec, and recombinant human interleukin-2 was obtained from Prometheus. Latrunculin A was obtained from Cayman Chemical, ML-7 from Santa Cruz Biotechnology, Inc., and 2-APB from Abcam.

Hu K.H, & Butte M.J. (2016). T cell activation requires force generation. The Journal of Cell Biology, 213(5), 535-542.

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Stimulation of Alveolar Macrophages with LPS and Luteolin

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For the experiments described in the manuscript, soluble stimuli were used at the following concentrations: lipopolysaccharide (LPS, InvivoGen) 500 ng/ml, CpG (InvivoGen) 100 nM, IL-1ß (ThermoFisher) 200 ng/ml, anti-CD40 (clone 1C10, Biolegend) 1 µg/ml, PMA (Sigma) 10⁻⁷M, ionomycin (Sigma) 1 μg/ml. For plate coating, anti-CD3ε (clone 145-2C11, Biolegend) and anti-CD28 (clone 37.51, Biolegend) were used at a concentration of 2 µg/ml, at and anti-IgM F(ab’)₂ (Jackson Immunoresearch Laboratories) at 10 µg/ml. For the stimulation of in vitro cultured alveolar macrophages, cells were collected using 4 mM EDTA in PBS and counted. 1 × 10⁵ cells per well were plated in an untreated 96-well plate overnight and stimulated on the consecutive day with 400 ng/mL LPS and/or 100 µM luteolin (Sigma-Aldrich). At the end of the stimulation supernatant was carefully removed and frozen for ELISA analysis. Cells were detached using 4 mM EDTA in PBS, washed once and stained with 1:4000 SytoxRed in PBS 2% FCS before flow cytometric analysis.

Tortola L., Piattini F., Hausmann A., Ampenberger F., Rosenwald E., Heer S., Hardt W.D., Rülicke T., Kisielow J, & Kopf M. (2022). KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo. Mucosal Immunology, 15(4), 656-667.

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Isolation and in vitro Stimulation of Naive CD8+ T Cells

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Naive OT-1 or P14 cells, respectively, from spleen were purified using naive CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec) and subsequently separated by AutoMACS (Miltenyi Biotec). Purity of the cells (95%) was confirmed by flow cytometry. For in vitro stimulation, 2×10⁵ naive CD8⁺ T cells were resuspended in D-MEM supplemented with 10% FCS (fetal calf serum), 2 mM GlutaMAX (GIBCO), 10 mM HEPES (GIBCO), 100 U/mL Penicillin/Streptomycin (GIBCO), and 50 μM 2-Mercaptoethanol and stimulated with anti-CD3 (clone 145.2C11, Biolegend), anti-CD28 (clone 37.51, Biolegend) (1 mg/ml) and recombinant murine IL-12 (Peprotech) in 96-well plates pre-coated with 100 μg/mL goat anti-hamster IgG (H+L) secondary antibody (Jackson Immunoresearch).

Page N., Klimek B., De Roo M., Steinbach K., Soldati H., Lemeille S., Wagner I., Kreutzfeldt M., Di Liberto G., Vincenti I., Lingner T., Salinas G., Brück W., Simons M., Murr R., Kaye J., Zehn D., Pinschewer D.D, & Merkler D. (2018). Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8+ T Cells. Immunity, 48(5), 937-950.e8.

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Isolation and Activation of Naïve CD8+ T Cells

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CD8⁺CD44^– T cells were purified from spleen and peripheral lymph nodes using the MojoSort™ Mouse CD8 Naïve T-Cell Isolation Kit (BioLegend, #480044) followed by CD25-depletion using the mouse CD25 MicroBead Kit (Miltenyl Biotec, #130-091-072), all according to manufacturers’ protocols. Naïve CD8⁺ T cells (1 × 10⁶/mL) were activated with plate-bound anti-CD3 (clone 145-2C11, 0.25 μg/ml, BioLegend) and anti-CD28 (clone 37.51, 0.5 μg/ml, BioLegend) in T-cell medium (high glucose DMEM, 10% FBS, penicillin/streptomycin, non-essential amino acids, HEPES, L-glutamate and sodium pyruvate) at 37°C in 10% CO₂ for 2 days. Cells were then removed from CD3/CD28 activation and re-cultured (5 × 10⁵/ml) in differentiation medium (T-cell medium supplemented with 10 IU/ml rhIL-2 from NCI) at 37°C in 10% CO₂. Cells were then sub-cultured every day in differentiation medium at 5 × 10⁵/ml each sub-culture. On day 5, cells were harvested and used for Western blots, ChIP-qPCR, qPCR or adoptive T-cell transfer.

Le P.T., Ha N., Tran N.K., Newman A.G., Esselen K.M., Dalrymple J.L., Schmelz E.M., Bhandoola A., Xue H.H., Singh P.B, & Thai T.H. (2021). Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence. Frontiers in Immunology, 12, 738958.

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