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Anti mouse cd28

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The Anti-mouse CD28 is a laboratory reagent designed for use in flow cytometry and other immunological applications. It functions as an antibody that specifically binds to the CD28 molecule expressed on the surface of mouse T cells. CD28 is a co-stimulatory receptor that plays a crucial role in the activation and proliferation of T cells. This reagent can be used to investigate the function and behavior of mouse T cells in various experimental settings.

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Lab products found in correlation

Anti mouse cd3e, by BD (8 mentions) Anti mouse cd3, by BD (8 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Cd8a microbeads, by Miltenyi Biotec (3 mentions) Hil 2, by Roche (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) E g7 ova cell, by American Type Culture Collection (2 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (2 mentions) Anti mouse gzmb, by Thermo Fisher Scientific (2 mentions) Granzyme b mouse elisa kit, by Thermo Fisher Scientific (2 mentions) Cd4 t cells, by Miltenyi Biotec (2 mentions) Cd11b cells, by Miltenyi Biotec (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Anti mouse cd4 microbeads, by Miltenyi Biotec (2 mentions) Recombinant il 6, by R&D Systems (2 mentions) Anti ifn γ, by BD (2 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-CD28 (379 citations) Hamster anti-mouse CD28 (8 citations) Mouse anti-human CD28 (6 citations) Soluble anti-mouse CD28 (4 citations) Mouse anti (2 citations) PE Mouse Anti-Human CD28 (2 citations) Mouse anti-human CD28 antibody (2 citations) NA/LE) anti-mouse CD28 mAb (2 citations) Anti-mouse CD28 mAb (clone 37.51, (2 citations) NA/LE mouse anti-human CD28 (2 citations)

28 protocols using anti mouse cd28

1

Quantifying Cytotoxic T Cell Responses Against Tumor Antigens

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EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS and 25mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145–2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×106 cells/ml in RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, and 30U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to uncoated plates. On day 5, 1 × 106 activated OT1 T cells were coincubated with 1×106 EL4 or EG7-OVA cells for 2 hours at 37 °C and stained for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience, 88–8824-00). To measure GzmB activity inside target cells, we coincubated activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at various T cell to target cell ratios and stained using GranToxiLux Kit (OncoImmunin, GTL702–8). To measure secretory GzmB, we collected coculture supernatant of OT1 with target cells and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience, BMS6029).
Mac Q.D., Mathews D.V., Kahla J.A., Stoffers C.M., Delmas O.M., Holt B.A., Adams A.B, & Kwong G.A. (2019). Non-invasive early detection of acute transplant rejection via nanosensors of granzyme-B activity. Nature biomedical engineering, 3(4), 281-291.
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2

Isolation and Activation of CD8+ T Cells

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Spleens from P14, pmel, or OT1 TCR transgenic mice (Jackson Labs) were dissociated in R10 media (RPMI 1640 (Gibco) + 10% FBS (Gibco) + 1% pen/strep (Gibco)), and red blood cells were lysed using RBC Lysis Buffer (Biolegend). CD8+ T cells were isolated using a CD8a+ T Cell Isolation Kit (Miltenyi Biotec). For pMHC liposome activation in vitro, T cells were cultured at 2×106 cells/ml in T cell media (R10 supplemented with 1X non-essential amino acids (Gibco) + 1 mM sodium pyruvate (Gibco) + 0.05 mM 2-mercaptoethanol (Sigma)), and liposomes were added at 1 μg/ml. For antibody-activated controls, T cells were cultured in T cell media supplemented with soluble anti-mouse CD28 (2 μg/ml, Clone: 37.51, BD Pharmingen) and 30 U/ml rhIL-2 (Roche) at 2×106 cells/ml in wells coated with anti-mouse CD3e (1 μg/ml, Clone: 145–2C11, BD Pharmingen).
Dahotre S.N., Romanov A.M., Su F.Y, & Kwong G.A. (2021). Synthetic Antigen-Presenting Cells for Adoptive T Cell Therapy. Advanced therapeutics, 4(8), 2100034.
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3

CD4+ T Cell Isolation and Stimulation

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Following red blood lysis with ACK lysis buffer, CD4+ T cells (Miltenyi Biotec) and CD11b+ cells (Miltenyi Biotec) were isolated by positive selection from splenocytes. For anti-CD3/anti-CD28 stimulation, 100,000 CD4+ T cells/well were cultured for 3 days in complete RPMI media in round bottom plates coated with 0.1, 0.5 and 1μg/ml of purified hamster anti-mouse CD3 (BD) and anti-mouse CD28 (BD) (see Reagent or Resource in the Key Resources Table).
Hoghooghi V., Palmer A.L., Frederick A., Jiang Y., Merkens J.E., Balakrishnan A., Finlay T.M., Grubb A., Levy E., Gordon P., Jirik F.R., Nguyen M.D., Schuurmans C., Visser F., Dunn S.E, & Ousman S.S. (2020). Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis. Cell reports, 33(1), 108236.
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4

Cytotoxicity Assay of Activated T Cells

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B16-F10 cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo). CD8+ T cells were isolated from either OT1 or Pmel (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145-2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×106 cells/ml in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 30 U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to untreated culture flasks for expansion. Between day 4 to 6 after activation, activated T cells were washed before being coincubated with 3×104 B16 target cells at various T cell to effector cell ratios. After 48 hours, coculture supernatants were collected for LDH and GzmB measurements by the Pierce LDH Cytotoxicity Assay Kit (Thermo) and GzmB Mouse ELISA Kit (Thermo, BMS6029) respectively. To assess sensor activation during T cell killing, cocultured of T cells and target cells were spiked in with either αPD1-GS, αPD1 conjugated with control peptide (LQRIYK), and unconjugated αPD1. After 48 hours, fluorescence of coculture supernatant were measured using Cytation 5 plate reader (Biotek).
Mac Q.D., Sivakumar A., Phuengkham H., Xu C., Bowen J.R., Su F.Y., Stentz S.Z., Sim H., Harris A.M., Li T.T., Qiu P, & Kwong G.A. (2022). Urinary detection of early responses to checkpoint blockade and of resistance to it via protease-cleaved antibody-conjugated sensors. Nature biomedical engineering, 6(3), 310-324.
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5

Activated CD4+ T Cell Proliferation Assay

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Mouse lymph nodes were derived from BALB/c mice. T cells were isolated from lymphocytes using a naïve CD4+ T Cell isolation kit (Miltenyi Biotec, German) following the manufacturer's instructions. The proliferation of CD4+ T cells was detected using carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, USA) labeling according to the manufacturer's instructions. To activate naïve CD4+ T cells, naïve CD4+ T cells were stimulated with immobilized 5 μg/ml anti-mouse CD3 antibody and 1 μg/ml anti-mouse CD28 (BD Pharmingen, USA) for 2-3 days. Activated CD4+ T cells were then cocultured with BMMSCs from NOD or BALB/c mice at the ratio of 1 : 1 for 3 days. The proliferation of T cells was determined by loss of CFSE fluorescence.
Su Y., Gu Y., Wu R, & Wang H. (2018). Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome. Stem Cells International, 2018, 9837035.
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6

T Cell Activation Assay Protocol

Cited 1 time
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Ninety-six well culture plates (Sigma-Aldrich, St. Louis, USA) were coated with anti-mouse-CD3e (clone: 145-2C11, BD Biosciences) at 10 μg/ml and stored overnight at 4°C. As described in previous studies in more detail, after isolation, 2 × 105 CD4+CD62Lhigh and CD4+CD62Llow cells were coincubated with either 2 × 105 DX5+NKT cells or CD8+ T cells in 200 μl RPMI culture medium (Gibco, Paisley, UK) in either coated or uncoated wells [7 (link)]. For further stimulation, 5 μg/ml anti-mouse-CD28 (clone: 37.51, BD Biosciences) and 2000 IU/ml IL-2 (PeproTech, Rocky Hill, USA) were added [31 (link)]. Control cultures of CD4+CD62Lhigh, CD4+CD62Llow, DX5+NKT cells, and CD8+T cells only were incubated at 4 × 105 cells in 200 μl RPMI under the same conditions.
Werner J.M., Damian M., Farkas S.A., Schlitt H.J., Geissler E.K, & Hornung M. (2018). Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand. Journal of Immunology Research, 2018, 8175810.
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7

Quantifying Cytotoxic T Cell Responses Against Tumor Antigens

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EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS and 25mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145–2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×106 cells/ml in RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, and 30U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to uncoated plates. On day 5, 1 × 106 activated OT1 T cells were coincubated with 1×106 EL4 or EG7-OVA cells for 2 hours at 37 °C and stained for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience, 88–8824-00). To measure GzmB activity inside target cells, we coincubated activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at various T cell to target cell ratios and stained using GranToxiLux Kit (OncoImmunin, GTL702–8). To measure secretory GzmB, we collected coculture supernatant of OT1 with target cells and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience, BMS6029).
Mac Q.D., Mathews D.V., Kahla J.A., Stoffers C.M., Delmas O.M., Holt B.A., Adams A.B, & Kwong G.A. (2019). Non-invasive early detection of acute transplant rejection via nanosensors of granzyme-B activity. Nature biomedical engineering, 3(4), 281-291.
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8

Murine T-Cell Activation and Cytokine Production

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Male C57BL/6 mice, 6 to 7 weeks old, were obtained from Harlan Laboratories (Indianapolis, IN). Hamster antimouse CD3ε and antimouse CD28 were obtained from BD Biosciences (San Diego, CA). Rat affinity purified antimouse CD16/32, hamster PE-conjugated antimouse CD11c, rat FITC-conjugated antimouse MHC II, rat APC-conjugated antimouse F4/80, hamster PE-Cy7-conjugated antimouse CD3ε, and recombinant IL-12 and IL-23 were obtained from eBioscience (San Diego, CA). Goat APC-conjugated antimouse IL-23R and IL-17 and IL-22 enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems (Minneapolis, MN). Collagenase D was obtained from Roche Applied Science (Indianapolis, IN). Concanavalin A (ConA), ionomycin calcium salt, phorbol 12-myristate 13-acetate (PMA), and AhR inhibitor CH-223191 were obtained from Sigma-Aldrich (St. Louis, MO).
Rendon J.L., Li X., Brubaker A.L., Kovacs E.J., Gamelli R.L, & Choudhry M.A. (2014). The Role of Aryl Hydrocarbon Receptor in Interleukin-23-Dependent Restoration of Interleukin-22 Following Ethanol Exposure and Burn Injury. Annals of surgery, 259(3), 582-590.
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9

Th17 Cell Differentiation from Mouse CD4+ T Cells

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CD4+ T cells were isolated from mouse splenocytes (SPL) using anti-mouse CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The sorted CD4+ T cells were stimulated with plate-bound anti-mouse CD3 (BD Biosciences, CA, USA) and anti-mouse CD28 (BD) and cultured with anti-mouse IFN-γ (R&D Systems, MN, USA) and IL-4 (R&D Systems) to block Th1 and Th2 differentiation, respectively. Th17 differentiation was induced by culture with recombinant IL-6 (R&D Systems) and recombinant transforming growth factor-β1 (R&D Systems) for 3 days.
Choi S., Lee K., Jung H., Park N., Kang J., Nam K.H., Kim E.K., Ju J.H, & Kang K.Y. (2018). Kruppel-Like Factor 4 Positively Regulates Autoimmune Arthritis in Mouse Models and Rheumatoid Arthritis in Patients via Modulating Cell Survival and Inflammation Factors of Fibroblast-Like Synoviocyte. Frontiers in Immunology, 9, 1339.
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10

Isolation and Characterization of Regulatory T Cells

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The CD4+CD25+ Regulatory T cell Isolation Kit and CD8+ T cell Isolation Kit were purchased from Miltenyi Biotec (Auburn, CA). Anti-mouse CD45R/B220 antibody used for preparation of bone marrow-derived dendritic cells was purchased from BD Bioscience (San Diego, CA), while antibodies directed against CD4 (GK1.5), CD8 (Lyt-2), and HB-32 were a kind gift from Dr. Xu of the University of Alabama at Birmingham. Dynabeads coupled with anti-rat IgG antibodies were purchased from Invitrogen (Carlsbad, CA). IL-2, IL-4, DNFB and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mouse CD3e, anti-mouse CD28 and GM-CSF were purchased from BD Bioscience (San Diego, CA). Mouse-specific ELISA kits for TGF-β, IL-10, and IFNγ were purchased from eBioscience (San Diego, CA). The GSPs were obtained from the Kikkoman Corporation (Japan) and the chemical composition of this product has been described previously [25 (link), 26 (link)]. Experimental diet containing GSPs (0.5%, w/w) was prepared commercially in pellet form in the AIN76A-powdered control diet by TestDiet (Richmond, IN) using the GSPs that we provided.
Vaid M., Prasad R., Singh T, & Katiyar S.K. (2017). Dietary grape seed proanthocyanidins inactivate regulatory T cells by promoting NER-dependent DNA repair in dendritic cells in UVB-exposed skin. Oncotarget, 8(30), 49625-49636.
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