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Ova323 339

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OVA323–339 is a synthetic peptide used for research purposes. It corresponds to a portion of the OVA323–339 epitope, which is a well-known T cell epitope derived from the chicken ovalbumin protein. The product can be used in various immunological studies and applications.

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Lab products found in correlation

Mog35 55, by AnaSpec (3 mentions) Il 2 recombinant protein, by Thermo Fisher Scientific (2 mentions) Anti il 2 mab clone jes6 1, by Thermo Fisher Scientific (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Anti cd3 and anti cd28 antibody, by Thermo Fisher Scientific (1 mentions) Macs bead, by Miltenyi Biotec (1 mentions) Recombinant mouse cytokines, by R&D Systems (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Lcmv gp61 80, by AnaSpec (1 mentions) Ova257 264, by AnaSpec (1 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions) Liberase cl, by Roche (1 mentions) Anti mouse ifn γ, by Thermo Fisher Scientific (1 mentions) Optiprep density gradient medium, by Merck Group (1 mentions) Macs ls column, by Miltenyi Biotec (1 mentions) Lps from escherichia coli 055 b5, by Merck Group (1 mentions) H3 thymidine, by PerkinElmer (1 mentions) Anti cd3, by BD (1 mentions) H37ra, by BD (1 mentions) Siinfekl, by InvivoGen (1 mentions)

11 protocols using ova323 339

1

Tracking Antigen-Specific T Cell Recruitment to CNS

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To determine whether myelin specific T cells require antigen-specific APCs for recruitment to the CNS, EAE was actively induced using a combination of two antigenic peptides in 8–12 week old female recipient mice. 3×106 naïve 2D2-CFP and 3×106 naïve OTII-GFP CD4+ T cells were isolated separately by negative depletion using Dynal beads and co-injected into naive recipient mice. 24 hours later, an emulsion of 200 μg MOG35-55 only, 10 μg OVA323-339 only, or 200 μg MOG35-55 and 10 μg OVA323-339 combined (Anaspec, San Jose, CA, USA) with CFA (8 mg/mL H37RA and IFA; Difco Laboratories, Detroit, MI, USA) was injected s.c. bilaterally on the lower back of the recipient mice. Control mice were injected s.c. bilaterally with an emulsion of PBS and CFA. 100 ng PTx (List Biological Laboratories, Inc, Campbell, CA, USA) was administered i.p. on days 0, 1 and 2 to induce EAE.
Barkauskas D.S., Dorand R.D., Myers J.T., Evans T.A., Barkauskas K.J., Askew D., Purgert R, & Huang A.Y. (2015). Focal Transient CNS Vessel Leak Provides a Tissue Niche for Sequential Immune Cell Accumulation during the Asymptomatic Phase of EAE Induction. Experimental neurology, 266, 74-85.
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2

IL-2:anti-IL-2 mAb Complexes for Thymic Slices

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IL-2:anti-IL-2 mAb complexes were described previously26 (link),27 (link). Briefly, 1.5 μg IL-2 recombinant protein (eBioscience) was incubated with 7.5 μg of functional grade anti-IL-2 mAb (clone JES6-1, eBioscience) for 30 min at 37 °C. Complexes were added directly to thymic slices (10 μl/slice) daily over the course of the experiment. Mouse MOG 35–55 and chicken OVA 323–339 peptides were purchased from Anaspec. Peptide was overlaid on thymic slices for 10–30 min after thymocyte migration into the slice.
Weist B.M., Kurd N., Boussier J., Chan S.W, & Robey E.A. (2015). Thymic regulatory T cell niche size is dictated by limiting interleukin 2 from antigen-bearing dendritic cells and feedback competition. Nature immunology, 16(6), 635-641.
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3

IL-2:anti-IL-2 mAb Complexes for Thymic Slices

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IL-2:anti-IL-2 mAb complexes were described previously26 (link),27 (link). Briefly, 1.5 μg IL-2 recombinant protein (eBioscience) was incubated with 7.5 μg of functional grade anti-IL-2 mAb (clone JES6-1, eBioscience) for 30 min at 37 °C. Complexes were added directly to thymic slices (10 μl/slice) daily over the course of the experiment. Mouse MOG 35–55 and chicken OVA 323–339 peptides were purchased from Anaspec. Peptide was overlaid on thymic slices for 10–30 min after thymocyte migration into the slice.
Weist B.M., Kurd N., Boussier J., Chan S.W, & Robey E.A. (2015). Thymic regulatory T cell niche size is dictated by limiting interleukin 2 from antigen-bearing dendritic cells and feedback competition. Nature immunology, 16(6), 635-641.
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4

In Vitro T Cell Differentiation

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Total splenocytes or naive CD4+/CD62L+ cells were incubated for 3 days in the presence of IL-2 (50 units/mL), IL-12(10 ng/mL), and anti-IL4(5 μg/mL) for Th1 cell differentiation; IL-2 (50 units/mL), IL-4 (10 ng/mL), and anti-IFNγ (5 μg/mL) for Th2 cell differentiation; or IL-6(40 ng/mL), TGFβ(5 ng/mL), IL-23(10 ng/mL), anti-IL4(5 μg/mL) and anti-IFNγ (5 μg/mL) for Th17 cell differentiation. All recombinant mouse cytokines were purchased from R&D Systems, Inc (San Diego, CA) and eBioscience. OT-II and DO11.10 splenocytes were activated by chicken ovalbumin peptide, OVA (323–339), (AnaSpec, Fremont, CA) and naïve CD4+/CD62L+ T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies from eBioscience, Inc. Naïve CD4+/CD62L+ T cells were isolated by MACS beads from Miltenyi Biotec (Auburn, CA).
Way E.E., Trevejo-Nunez G., Kane L.P., Steiner B.H., Puri K.D., Kolls J.K, & Chen K. (2016). Dose-Dependent Suppression of Cytokine production from T cells by a Novel Phosphoinositide 3-Kinase Delta Inhibitor. Scientific Reports, 6, 30384.
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5

Antigen-Specific T Cell Activation by DCs

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For DC transfer experiments, splenic DCs were isolated as described above from mice subcutaneously injected with 1 × 106 B16 melanoma cells that constitutively secrete FMS-like tyrosine kinase 3 ligand (Flt3L)58 (link) 10 days prior to harvest. Cells were resuspended at 107 cells/ml and incubated with 10 μM OVA323–339, LCMV-GP61–80, OVA257–264, or LCMV276–286 peptides (Anaspec) in RPMI + 10% FBS, for 30 min at 37 °C. For cell labelling, CFSE or CTV (ThermoFisher Scientific) was added to a final concentration of 2 μM during the last 5 or 20 minutes of incubation, respectively. Cells were washed three times in RPMI + 10% FBS and resuspended at 2 × 107 cells/ml in PBS supplemented with 0.4 μg ml−1 LPS (Sigma-Aldrich). DCs were injected (5 × 105 cells in 25 μl) subcutaneously into the hind footpads. For CD4+ T cell and CD8+ T cell transfer experiments, T cells isolated as described above were injected intravenously in 100 μl PBS per mouse.
Nakandakari-Higa S., Canesso M.C., Walker S., Chudnovskiy A., Jacobsen J.T., Bilanovic J., Parigi S.M., Fiedorczuk K., Fuchs E., Bilate A.M., Pasqual G., Mucida D., Pritykin Y, & Victora G.D. (2023). Universal recording of cell–cell contacts in vivo for interaction-based transcriptomics. bioRxiv.
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6

Th2 Cell Differentiation from Mouse T Cells

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Single-cell suspensions from BT-II or OT-II mouse lymph nodes were incubated with anti-CD4 MicroBeads isolated on a MACS LS column (Miltenyi Biotec, Singapore). Splenic dendritic cells (DCs) were digested with Liberase Cl (Roche, Switzerland) at 37°C for 30 min and isolated by centrifugation over OptiPrep Density Gradient Medium (Sigma-Aldrich, St Louis, MO), incubated with anti-mouse CD11c MicroBeads, and passed through a MACS LS column (Miltenyi Biotec). BT-II and OT-II CD4 T cells and splenic DCs were cocultured in 48-well plates with 10 μg/ml Blo t 555–70 peptide (IIRELDVVCAMIEGAQ) or OVA323–339, respectively (AnaSpec, Fremont, CA); 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ)1; 20 μg/ml anti-mouse IFN-γ; 20 μg/ml anti-mouse IL-12/IL-23 p40 (eBioscience, ImmunoCell, Singapore); and 20 μg/ml mouse IFN-γ R1/CD119 Ab (R&D Systems, Minneapolis, MN). Cells were restimulated on days 3, 7, and 11 with Blo t 555–70, cytokines, and neutralizing Abs. Fresh DCs were added on day 7. Th2-polarized BT-II cells were harvested on day 14.
Chua Y.L., Liong K.H., Huang C.H., Wong H.S., Zhou Q., Ler S.S., Tang Y., Low C.P., Koh H.Y., Kuo I.C., Zhang Y., Wong W.S., Peh H.Y., Lim H.Y., Ge M.Q., Haczku A., Angeli V., MacAry P.A., Chua K.Y, & Kemeny D.M. (2016). Blomia tropicalis–Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13–Dependent Mechanism. Journal of immunology (Baltimore, Md. : 1950), 197(10), 3771-3781.
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7

OT-I T cell activation and transfer

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Spleens from OT-I, Rag−/− OT-I, or OT-II mice were harvested and processed into single cell suspension as described above. Splenocytes were activated in complete RPMI in the presence of 50 units/mL recombinant IL-2 and 1 μg/mL OT-I cognate peptide OVA257–264 (SIINFEKL) or OT-II cognate peptide OVA323–339 for four days (AnaSpec, Inc). Activated OT-I T cells were transferred into mice i.v. via tail vein.
Kaczanowska S., Beury D.W., Gopalan V., Tycko A.K., Qin H., Clements M.E., Drake J., Nwanze C., Murgai M., Rae Z., Ju W., Alexander K.A., Kline J., Contreras C.F., Wessel K.M., Patel S., Hannenhalli S., Kelly M, & Kaplan R.N. (2021). Genetically Engineered Myeloid Cells Rebalance the Core Immune Suppression Program in Metastasis. Cell, 184(8), 2033-2052.e21.
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8

Adoptive Transfer of OT-II T Cells

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Control (nontumor-bearing) OT-II mice (CD90.1+) and OT-II mice (CD90.1+) bearing Eμ-Myc lymphomas 14 days after transplant were euthanized, and CD4+ T cells were isolated from spleens as described. 1.5 × 106 CD4+ T cells were then adoptively transferred into sublethally irradiated congenic C57BL/6J CD90.2+ hosts via tail vein injection (JL Shepherd Mark 1, Model 68A CS-137 Irradiator; 600 RAD, <24 hours prior to tail vein injection). Mice were immediately injected intraperitoneally with 100 μg ovalbumin 323–339 peptide (OVA323–339, Anaspec) and 10 μg lipopolysaccharide (LPS, Sigma) in sterile PBS. Mice were euthanized after 96 hours, and CD90.1+ OT-II cells were analyzed via flow cytometry from single cell suspensions of splenocytes as described below.
Hesterberg R.S., Liu M., Elmarsafawi A.G., Koomen J.M., Welsh E.A., Hesterberg S.G., Ranatunga S., Yang C., Li W., Lawrence H.R., Rodriguez P.C., Berglund A.E, & Cleveland J.L. (2022). TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate. Cancer Immunology Research, 10(10), 1263-1279.
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9

Myeloid Cell-Mediated Modulation of T Cell Responses

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Myeloid cells (BM-MCs or BM-MCregs) were cultured with responder spleen cells from antigen-specific T cell receptor transgenic (TCR Tg; where antigen was either OVA323-339 or MOG35-55) or Foxp3EGFP mice as indicated. To assess T cell proliferation co-cultures were stimulated with anti-CD3 (BD Bioscience), T cell-receptor specific antigen MOG35-55 (Bio Matic) or T cell-receptor specific antigen OVA 323–339 (Anaspec). To assess the effects of myeloid cell activation, co-cultures were stimulated with LPS from Escherichia coli, 055:B5 (Sigma-Aldrich) for 96 hours, pulsed with (H3 thymidine) (Perkin Elmer Life Sciences or MP Biomedicals) in the last 18 hours, harvested and counted, data is expressed as counts per million (cpm) ± SEM [35 (link)].
VanGundy Z.C., Guerau-de-Arellano M., Baker J.D., Strange H.R., Olivo-Marston S., Muth D.C, & Papenfuss T.L. (2014). Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells. BMC Immunology, 15, 8.
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10

OVA Peptide Immunization in Mice

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A mixture of OVA peptides was administrated in a s.c. injection in mice anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). OVA257–264 (SIINFEKL) (InvivoGen, San Diego, CA, USA) is a class I (Kb)-restricted peptide epitope of OVA presented by the class I MHC molecule (CMH) H-2Kb. OVA323–339 (ISQAVHAAHAEINEAGR) (AnaSpec, San Jose, CA, USA) is an H-2b-restricted OVA class II epitope. 100 μg of each peptide emulsified with the same volume of IFA (Sigma-Aldrich) was administered. Controls were injected with PBS-IFA complexes. OVA peptides and PBS-IFA complexes were injected 2 h or 21 days post-first i.p. injection of MnTBAP (or PBS). Eight days post-OVA peptide injection, mice were sacrificed, and spleens were harvested to analyze OVA-specific T cell response.
Da Rocha S., Bigot J., Onodi F., Cosette J., Corre G., Poupiot J., Fenard D., Gjata B., Galy A, & Neildez-Nguyen T.M. (2019). Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer. Molecular Therapy. Methods & Clinical Development, 14, 285-299.
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