Ova peptide

Manufactured by GenScript

Sourced in United States

OVA peptide is a synthetic peptide that corresponds to a specific amino acid sequence. It is commonly used as a reference standard or control in various research and experimental applications.

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Lab products found in correlation

Spelling variants of this product

OVA peptide 323–339 (5 citations) OVA peptide (OVA 323–339) (2 citations) N4 OVA peptide (2 citations)

8 protocols using ova peptide

CD4+ T Cell Activation Assay

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Spleen and LNs of DO11.10 mice were harvested, digested in RPMI-1640 medium (GIBCO, Paisley, UK) containing liberase CI (0.42 mg/ml (1.67 U/ml), Roche) and DNase 1 (0.2 mg/ml (400 U/ml), Roche) and passed through a 40 µm cell strainer (BD Biosciences). Single-cell suspensions were incubated for 20 min with anti-CD4⁺-coupled magnetic beads and isolated using a MACS column and separator (all from Miltenyi Biotech, Gladbach, Germany). Isolated CD4⁺ T cells (purity > 90%) were stained with 2 µM CFSE (Invitrogen) for 12 min at 37°. Subsequently, T cells were co-cultured for 3 days with FACS-sorted DCs, at a T cell:DC ratio of 5∶1, in presence of 100 µM OVA peptide (residues 323-339, GenScript, Piscataway, NJ, USA). Co-cultures were performed in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 50 µM β-mercaptoethanol (Sigma), 15 mM HEPES (GIBCO), 1 mM Na-pyruvate (GIBCO), antibiotic/antimycotic solution (Fluka, Buchs, Switzerland) and 2 mM L-glutamine (Fluka).

Aebischer D., Willrodt A.H, & Halin C. (2014). Oxazolone-Induced Contact Hypersensitivity Reduces Lymphatic Drainage but Enhances the Induction of Adaptive Immunity. PLoS ONE, 9(6), e99297.

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OT-I Splenocyte Activation and Glucose Uptake

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Splenocytes from OT-I mice were activated with OVA peptide 10 μg/ml (GenScript)/APCs, recombinant huIL-2 100 units/mL (Thermo Fisher) in complete RPMI media (Gibco). In some experiments, cells were labeled with CFSE 5 μM (TONBO) to track cell division. Some cells were incubated with iPI3Kδ 10 μg/ml. Cells were collected after 3 or 4 days, washed with FACS buffer and stained for CD8 followed by fixation and permeabilization and staining of pS6 and TCF1. Glucose uptake was measured after collection by washing cells two times, and incubating cells in 2-NBDG³ 100 μM (Cayman) at 45 mins, 37C in the dark.

Wang P.H., Washburn R., Maniar R., Mu M., Ringham O., Kratchmarov R., Henick B.S, & Reiner S.L. (2022). Promoting TCF1+ T-cell self-renewal to improve PD-1 blockade. Journal of immunology (Baltimore, Md. : 1950), 209(4), 660-664.

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Quantifying T Cell Proliferation Assay

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For T cell proliferation assays, MACS sorted cDC from rhMPV (-WT or -ΔG) and mock-infected mice (10⁵ cells/well) were cultured for 5 days with CD4+ T cells from DO11.10 mice (2x10⁵ cells/well) in 96-well round bottom microtiter plates. cDC were loaded with 10 μg/mL of OVA peptide (323–339, Genscript, Piscataway, NJ) 2h prior to co-culture with T-cells. The CD4+ T cells were tagged with CFSE prior to culture and after 5 days of co-culture, cells were collected, washed and analyzed by FACS for T-cell proliferation, as determined by increase in CFSE low positive cells (36 (link)).

Velayutham T.S., Ivanciuc T., Garofalo R.P, & Casola A. (2022). Role of human metapneumovirus glycoprotein G in modulation of immune responses. Frontiers in Immunology, 13, 962925.

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Adoptive Transfer of OT-II and OT-I Cells

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CD4⁺ OT-II and CD8⁺ OT-I cells were isolated as previously described. A mixture of 1 × 10⁶ CD4⁺ OT-II and 4 × 10⁵ CD8⁺ OT-I cells was injected i.v. into recipient wild-type mice. One day later, mice were immunized s.c. with 50 μl of the indicated NP into both flanks. Spleens were harvested 5 days p.i. Tissues were processed to yield a single cell suspension. After lysis of red blood cells, cell suspensions were washed and filtered through a 70 μm cell strainer. Cells were then incubated O.N. at 37°C, 5% CO₂ in RPMI (Biological Industries) and stimulated with PMA/Ionomycin activation cocktail (1:2000, BioLegend), Brefeldin A (0.5 μg/ml, BioLegend) and OVA peptide (323-339, 20 μg/ml, GenScript). On the following day cells were stained for extracellular TCRβ, CD4, CD45.1, CD44 and intracellular IFNγ and IL-17A according to the guidelines of the Cytofix/perm kit (BD). Cells were analyzed by flow cytometry.

Curato C., Bernshtein B., Zupancič E., Dufner A., Jaitin D., Giladi A., David E., Chappell-Maor L., Leshkowitz D., Knobeloch K.P., Amit I., Florindo H.F, & Jung S. (2019). DC Respond to Cognate T Cell Interaction in the Antigen-Challenged Lymph Node. Frontiers in Immunology, 10, 863.

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Isolation of Antigen-Loaded Exosomes

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BMDCs were activated with LPS, loaded with either 1 µM OVA peptide (GenScript) or 1 µM OVA peptide-FITC (Eurogentec), and cultured in exosome-depleted medium for 24 h. Exosomes were then purified using serial centrifugations: 300 g for 10 min, 2,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min (twice). Pellet was only kept for the last two ultracentrifugations and resuspended in small volumes of PBS. For FACS analysis, exosomes were coupled to Aldehyde-Sulfate Latex beads 4% wt/vol 9 µm (Life Technologies; 10:1 ratio), and identified as CD63⁺. In indicated experiments, exosomes were incubated for 12 h with LNSCs on a plate shaker at 37°C.

Dubrot J., Duraes F.V., Potin L., Capotosti F., Brighouse D., Suter T., LeibundGut-Landmann S., Garbi N., Reith W., Swartz M.A, & Hugues S. (2014). Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance. The Journal of Experimental Medicine, 211(6), 1153-1166.

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OT-1 T Cell Cytotoxicity Assay

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Splenocytes were isolated from OT-1 mice (4–6 weeks old) and activated with 100 nM OVA peptide (257–264; GenScript) and 50 U ml^–1 h IL-2 (PeproTech) for 48 h. After 48 h, fresh medium with 50 U ml^–1 IL-2 was added to the culture and incubated for an additional 24 h. Cells were then collected, replated and expanded at 1 × 10⁶ cells per ml.
For the specific cytotoxicity assay, tumor cells (target) were plated at 50,000 cells per well in a 24-well plate. OT-1 T cells (effector) were then added at a 1:5 target:effector ratio. Where indicated, GPCR agonists were added at the following concentrations: 1 μM 16,16-dimethyl PGE₂ (Tocris), 5 μM dobutamine hydrochloride (Tocris) and 5 μM CGS-21680 hydrochloride (Tocris). The coculture was left for 36 h, and cell viability was assessed by flow cytometric staining with Zombie Aqua viability dye (BioLegend).

, & Phillippy B.Q. (1998). Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds. Plant Physiology, 116(1), 291-297.

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Co-culture of BMDMs and Antigen-Specific T Cells

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BMDMs and T cells were co-cultured as described previously (Gojkovic et al, 2021 (link)). Briefly, for BMDM culture, bone marrow cells from the femur and tibia of WT and Gsdmd^−/− male mice (8−12 weeks) were cultured for 6 days in DMEM supplemented with 10% fetal bovine serum (FBS) and 10% L929 conditioned medium. The primary BMDMs were plated on 96-well round bottom plates (50,000 cells/well) and grown for 2 h. Subsequently, the BMDMs were pre-stimulated with 100 ng/ml LPS (Enzolife Sciences, ALX-581-0) for 24 h, treated with nigericin (Invivogen, tlrl-nig-5) for 15 min, washed thrice with PBS, and cultured in DMEM supplemented with 10% FBS and 100 ng/ml OVA peptide (GenScript, RP10611) for 6 h. The expression of antigen presentation-associated genes in BMDMs was analyzed using flow cytometry. Naive CD8⁺ T cells were isolated from the spleen and lymph nodes of OT-I mice (8−12 weeks) using a cell isolation kit (STEMCELL, 19858). For co-culturing, 20,000 T cells were added to the BMDMs in a 96-well plate after 3 days, and the activation and function of antigen-specific T cells were detected using flow cytometry.

Ma C., Huang J., Jiang Y., Liu L., Wang N., Huang S., Li H., Zhang X., Wen S., Wang B, & Yang S. (2024). Gasdermin D in macrophages drives orchitis by regulating inflammation and antigen presentation processes. EMBO Molecular Medicine, 16(2), 361-385.

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T-cell Cytokine Response Assay

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DO11.10⁺/CD4⁺ T-cells and BMDCs were co-cultured as described in the section “Stimulation of carboxyfluorescein succinimidyl ester (CFSE)-labeled OVA-specific T-cells” with or without 20 μg/mL OVA peptide (sequence 323–339; GenScript) for 72 hours. On day 3, Brefeldin A (20 μg/mL; eBioscience) was added to the cultures for 2 hours. At the end of the culture period, cells were incubated with antibodies against CD4 and the DO11.10 T-cell receptor (KJ-126) for 20 minutes on ice prior to fixation in 1% formalin solution for 20 minutes. Cells were incubated in permeabilization buffer (PBS + 0.1% saponin +10% FCS) for 5 minutes at room temperature before the following anti-cytokine antibodies were added for 30 minutes: tumor necrosis factor (TNF)α-eF450, interferon (IFN)γ-eF450, interleukin (IL)10-PE, IL5-PE, IL17-PE-Cy7, and IL4-APC (all eBioscience).

Seydoux E., Rothen-Rutishauser B., Nita I.M., Balog S., Gazdhar A., Stumbles P.A., Petri-Fink A., Blank F, & von Garnier C. (2014). Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation. International Journal of Nanomedicine, 9, 3885-3902.

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