Ova257 264

Manufactured by AnaSpec

OVA257-264 is a fluorescent-labeled peptide synthesized by AnaSpec. It is designed for use in immunological studies. The product consists of a peptide sequence coupled to a fluorescent dye, enabling its detection and quantification.

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Lab products found in correlation

Lcmv gp61 80, by AnaSpec (2 mentions) 70μm nylon mesh strainer, by Thermo Fisher Scientific (1 mentions) C57bl 6 tg, by Jackson ImmunoResearch (1 mentions) Lcmv gp33 41, by AnaSpec (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions) Anti ifnar, by BioXCell (1 mentions) Matrigel, by Corning (1 mentions) Ova323 339 peptide, by AnaSpec (1 mentions) Ova323 339, by AnaSpec (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Vibratome, by Leica (1 mentions) Cell culture insert, by BD (1 mentions) Ova323 339 peptide, by InvivoGen (1 mentions)

6 protocols using ova257 264

Activation of Murine T Cells with Rexinoids

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Spleens were harvested from B6.Cg-Tcra^tm1MomTg(TcrLCMV)327Sdz/TacMmjax (P14), B6.Cg-Ptprc^aPepc^bTg(TcrLCMV)1Aox/PpmJ (SMARTA), or C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) transgenic mice (Jackson Labs) and mechanically dissociated into a single cell suspension using a 70μm nylon mesh strainer (Fisherbrand). All mouse experiments were conducted under institutional care and use committee approval. T cells were stimulated with 1μg/mL of appropriate viral peptide (LCMV gp_33-41, LCMV gp_61-80, or OVA_257-264, respectively) (GenScript; Anaspec). Cells were cultured in a 96-well plate for 8 days using RPMI complete medium (10% FBS, 1% PSG 100X) supplemented with 2.5x10^-5 μg/μL IL-2 (Gibco) and 100nM of indicated rexinoid treatment or ATRA in a final volume of 200μL. 8 day treatment timeframe was determined using a time course assay (Supplementary Figure 1). Fresh culture medium with IL-2 and rexinoid or ATRA was replaced every 48 hours. Vitamin A deficient media was made using charcoal-stripped FBS (ThermoFisher).

Manhas K.R., Marshall P.A., Wagner C.E., Jurutka P.W., Mancenido M.V., Debray H.Z, & Blattman J.N. (2022). Rexinoids Modulate Effector T Cell Expression of Mucosal Homing Markers CCR9 and α4β7 Integrin and Direct Their Migration In Vitro. Frontiers in Immunology, 13, 746484.

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Tumor Immunotherapy via Adoptive T Cell Transfer

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One day after naive CD8⁺ T cell transfer, the flank of mice was shaved and B16F10 or B16-OVA cells (2.5–3 × 10⁵) were mixed with Matrigel (Corning, final concentration 5 mg/ml) and injected subcutaneously (s.c.). Tumor volumes were estimated by measuring the tumor size in three dimensions (length, width, and height) using a caliper. The tumor volume was calculated according to the formula (π/6 × length × width × height). Mice were sacrificed at the indicated time points or when the estimated tumor volume reached >1000 mm³ (endpoint) and the weight of the excised tumor mass was determined.
For tumor vaccination, mice were injected subcutaneously (beside the tumor) with 50 μg/mouse poly(I:C) (Invivogen) together with antigenic peptides (for Pmel-1, gp100_25-33; for OT-1, OVA_257-264) at 10 μg/mouse (peptides were purchased from Anaspec). The control (CON) group mice were injected with PBS in same volume.
For checkpoint blockade, mice were injected with rat anti-mouse PD-L1 or isotype control (BioXcell) (200 μg per injection, i.p.) once every 4 days for a total of three injections.
Anti-IFNAR (BioXcell) (500 μg/injection, i.p.) was given twice when indicated. The first time was given together with tumor vaccine and the second injection was administrated 2 days later.

Li G., Srinivasan S., Wang L., Ma C., Guo K., Xiao W., Liao W., Mishra S., Zhang X., Qiu Y., Lu Q., Liu Y, & Zhang N. (2022). TGF-β-dependent lymphoid tissue residency of stem-like T cells limits response to tumor vaccine. Nature Communications, 13, 6043.

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Ovalbumin and CpG Immunization Protocol

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OVA was purchased from Worthington. OVA-Al-647 and OVA-Al 488 was purchased from Molecular probes. OVA(257–264) and OVA(323–339) peptide epitopes were purchased from Anaspec Inc. ODN1826 (CpG; TCC ATG ACG TTC CTG ACG TT) is a 20-mer with a nuclease-resistant phosphorothioate backbone and was purchased from Invivogen. ODN1826 is a class B CpG that has potent Th1 skewing properties in mice. Mice were immunized either subcutaneous in the footpad (20 µl volume) or at the tail basis (100 µl volume) with a mixture of OVA (100 µg/ml) and CpG (100 µg/ml).

De Koker S., Van Hoecke L., De Beuckelaer A., Roose K., Deswarte K., Willart M.A., Bogaert P., Naessens T., De Geest B.G., Saelens X., Lambrecht B.N, & Grooten J. (2017). Inflammatory monocytes regulate Th1 oriented immunity to CpG adjuvanted protein vaccines through production of IL-12. Scientific Reports, 7, 5986.

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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 OVA^323–339, LCMV-GP^61–80, OVA^257–264, or LCMV^276–286 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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Studying T Cell Activation in Thymic Slices

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For F5 in vivo peptide injection studies, F5 TCR transgenic mice were injected intraperitoneally with 50 nmol of Influenza NP_366-374 Strain A/NT/60/68 peptide (AnaSpec, Fremont, CA) in 200 μl of PBS or PBS only for controls. A second injection was performed 24 hr after the first. Thymocytes were collected at 48 hr after the initial injection.
For F5 and HY thymic slice assays, tissue preparation was performed as previously described (Dzhagalov et al., 2012 (link), 2013 (link)). In brief, thymic lobes were embedded in 4% GTG-NuSieve Low-melt Agarose (Lonza, Walkersville, MD) in HBSS. 500 μm slices were cut by Vibratome (1000 Plus, Leica, Buffalo Grove, IL) and placed in 0.4 μm Cell Culture Inserts (BD Biosciences) in 6-well plates containing 1 ml of RPMI media. TCR-specific (NP_366-374 Strain A/NT/60/68 and Smcy HY_738-746, AnaSpec) and control (NP_366-374 Strain A/PR/8/35 and OVA_257-264, AnaSpec) peptides were diluted to 100 ng/ml in RPMI media and 1 ml volume was added to the slices. Slices were incubated at 37°C in a plastic bag filled with 80% O₂ + 15% N₂ + 5% CO₂ (Blood Gas, Praxair, Danbury, CT) for 30 min. The peptide was then removed and slices were further incubated for the indicated times. Thymic slices were dissociated to create single cell suspensions and cell death was assessed by Cleaved Caspase 3 staining as described above.

Burger M.L., Leung K.K., Bennett M.J, & Winoto A. (2014). T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity. eLife, 3, e03468.

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Assessing T cell suppressive function

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To assess the T cell suppressive function of CD11b⁺Ly6C^hiLy6G⁺ cells, splenocytes from OT-I or OT-II transgenic mice (2×10⁵ cells/well) were cultured in RPMI 1640 complete medium in the presence or absence of splenic CD11b⁺Ly6C^hiLy6G⁺ cells sorted from young and old mice at a 1:1 ratio, as previously described [26]. The co-cultures were then treated with 2.5 μg/ml of OVA_257–264(AnaSpec) or OVA_323–339 peptide (InvivoGen) for 72 h, respectively. CD8⁺ and CD4⁺ T cell proliferation was assessed using a FITC BrdU flow cytometry kit (Invitrogen). In brief, following incubation with BrdU (10 μM) for 20 h, cells were stained with PE-labeled anti-mouse CD8 antibody (Invitrogen) or APC-labeled anti-mouse CD4 antibody (Invitrogen), fixed, and permeabilized. Cells were then treated with DNase I for 1 h at 37°C, followed by staining with FITC-conjugated anti-BrdU antibody. Data were acquired using a BD LSR II flow cytometry and analyzed using FlowJo software v10.

Li Z., Zhao Y., Chen Z., Katz J., Michalek S.M., Li Y, & Zhang P. (2021). Age-related expansion and increased osteoclastogenic potential of myeloid-derived suppressor cells. Molecular immunology, 137, 187-200.

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