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Ova257 264 peptide

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OVA257-264 peptide is a synthetic peptide representing amino acid residues 257-264 of the chicken ovalbumin protein. The sequence is SIINFEKL. This peptide can be used in various research applications.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (1 mentions) El4 cells, by American Type Culture Collection (1 mentions) Easysep mouse cd8 t cell enrichment kit, by STEMCELL (1 mentions) Vital exclusion dye, by Thermo Fisher Scientific (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Easysep mouse pan dc enrichment kit 2, by STEMCELL (1 mentions) Celltrace violet dye, by Thermo Fisher Scientific (1 mentions) Anti cd3, by BioLegend (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Anti cd28, by BioLegend (1 mentions) Wash buffer, by BioLegend (1 mentions) Np366 374 peptide, by AnaSpec (1 mentions) Ionomycin, by Merck Group (1 mentions) Monensin, by BD (1 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Il 15, by Thermo Fisher Scientific (1 mentions) Magnetic bead, by Miltenyi Biotec (1 mentions) Gm csf, by Amgen (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

12 protocols using ova257 264 peptide

1

OT-I Mouse CD8+ T Cell Activation

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Exhausted T cells were prepared as described.(17 (link)) CD8+ T cells were purified from C57BL/6-Tg(TcraTcrb)1100Mjb/J (commonly known as OT-I) mouse splenocytes with magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were cultured at a final density of 5 × 105/mL in complete medium (RPMI 1640, 10% FBS, 1% 2 mM l-glutamine, 1% 1 M HEPES, 1% 100 mM sodium pyruvate, 1% nonessential amino acids, 1% penicillin–streptomycin, 0.05 mM beta-mercaptoethanol) with interleukin (IL)-15 (5 ng/mL; Peprotech, East Windsor, NJ) and IL-7 (5 ng/mL; Peprotech) with 10 ng/mL OVA(257–264) peptide (Anaspec, Fremont, CA).
Cells were repetitively stimulated with 10 ng/mL OVA(257–264) peptide daily for 5 to 7 days. Cells were checked daily, and when confluent, split, and cultured with fresh complete medium containing cytokines. For flow cytometry analysis, cells were first incubated with anti-Fc receptor antibody clone 2.4G2 (1 μg per well) for 30 minutes at room temperature. The indicated concentration of PE-conjugated PD-1 mAb 1A12, RMP1-14, or RMP1-30 or isotype control mAb was added and incubated for 30 minutes at 4°C. Cells were washed twice and binding was analyzed by flow cytometry.
Bu M.T., Yuan L., Klee A.N, & Freeman G.J. (2022). A Comparison of Murine PD-1 and PD-L1 Monoclonal Antibodies. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 41(4), 202-209.
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2

Ibrutinib Modulates Exhausted T Cell Cytotoxicity

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Murine T cells were induced to be exhausted as described above. Exhausted T cells were either treated with 1 μM ibrutinib or DMSO from day 5 to day 8 for a total of 3 days. On day 8, cells were washed thoroughly and rested for 24 hours before being seeded together with AE-17 tumor cells. For the co-culturing, AE-17 cells were resuspended with cell culture medium (RPMI 1640 with 10% FBS) at 106 cell/ml, then pulsed with 1μg/ml OVA(257-264) peptide (Anaspec). After being incubated for 1 hour at 37˚C, cells were labelled with CellTraceTM Far Red fluorescent dye (Invitrogen™, C34564) as specific target cells. Equal numbers of un-pulsed cells were labelled with CellTrace™ CFSE fluorescent dye (Invitrogen™, C34554) as non-specific control cells. For killing assay, 105 peptide-pulsed and un-pulsed AE-17 cells were seeded in 12-well plate and co-cultured with different ratios of T cells (Effector: Target ratio: 0:1, 0.5:1, 1:1, 3:1) for 18 hours. The next day, cells were harvested and stained for flow cytometry. The calculation of specific killing used the formula: % specific lysis = 100 –[Target/Non-target (in the presence of effector cells)]/[Mean of Target/Non-target (in the absence of effector cells)] x 100%.
Li L., Zhao M., Kiernan C.H., Castro Eiro M.D., van Meurs M., Brouwers-Haspels I., Wilmsen M.E., Grashof D.G., van de Werken H.J., Hendriks R.W., Mueller Y.M, & Katsikis P.D. (2023). Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK. Frontiers in Immunology, 14, 1201415.
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3

Cytotoxicity Assay of CD8+ T Cells

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ex vivo cytotoxic potential of CD8+ T cells was measured as previously described (45 (link)). Briefly, EL-4 cells (ATCC) were pulsed with 2 µM OVA257-264 peptide (AnaSpec) or no peptide for 1hr at 37°C. Peptide pulsed cells were stained with CFSE (Life Technologies) and unpulsed cells were stained with CellTrace Violet (Life Technologies) per manufacturers instructions. CD8+ T cells were enriched by negative selection using the EasySep Mouse CD8+ T cell enrichment kit (StemCell Technologies). The number of effector cells were determined by quantification of Kb/OVA+ CD8+ T cells and the appropriate numbers were added and the mixtures were incubated for 5 hrs at 37 °C. Following incubation, cells were stained with a vital exclusion dye (Life Technologies) to exclude dead cells. Fixed cells were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo v9.7.2 (Treestar). Specific lysis was calculated as 100 – [100 × ( % survival / average % survival in absence of effector cells)].
Provine N.M., Larocca R.A., Aid M., Penaloza-MacMaster P., Badamchi-Zadeh A., Borducchi E.N., Yates K.B., Abbink P., Kirilova M., Ng’ang’a D., Bramson J., Haining W.N, & Barouch D.H. (2016). Immediate Dysfunction of Vaccine-Elicited CD8+ T Cells Primed in the Absence of CD4+ T Cells. The Journal of Immunology Author Choice, 197(5), 1809-1822.
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4

TMAO-Mediated T Cell Suppression Assay

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For T cell suppression assay, we primed BMDM with 20% TCM in the presence of 300µM TMAO for 24hr. OT-I T cells (CD8+) were isolated from spleens of OT I mice using EasySep™ Mouse CD8+ T Cell Isolation Kit (Stem Cell Technologies, 19853A) and were labelled with CellTrace™ Violet dye (Invitrogen, C34557A) at a final concentration of 5µM. Dendritic cells (DCs) were isolated from spleens of wild type mice using EasySep™ Mouse Pan-DC Enrichment Kit II (Stem Cell Technologies, 19863). Co-cultures were set up in triplicates with BMDM primed as above, OT I T cells, and DCs in the presence of OVA257–264 peptide (Ana Spec Inc., AS-60193–1) at 0.5ng/ml for 3 days. DCs were added at 1:10 ratio to T cells. T cell proliferation was assessed by flow cytometry for dilution of CellTrace™ Violet stain on CD8+ T cells. In experiments using anti-CD3 and anti-CD28 induced T cell proliferation, CD8+ T cells were isolated from spleens of wild type mice and were co-cultured with BMDM primed as above in the presence of 0.1 µg of anti-CD3 (Biolegend, 100340) and anti-CD28 (Biolegend, 102116).
Mirji G., Worth A., Bhat S.A., Sayed M.E., Kannan T., Goldman A.R., Tang H.Y., Liu Q., Auslander N., Dang C.V., Abdel-Mohsen M., Kossenkov A., Stanger B.Z, & Shinde R.S. (2022). The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer. Science immunology, 7(75), eabn0704.
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5

Multiparametric Analysis of CD8+ T Cell Function

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Single cell suspension were stimulated with PMA (0.1 μg/ml) and Ionomycin (1 μg/ml) (Sigma) or OVA257-264 peptide (Anaspec, 1 μg/ml) or NP366-374 peptide (Anaspec,1 μg/ml) for 5h in the presence of 2μM monensin (BD Biosciences), and then stained with APC-CY7 conjugated anti-CD8. Cells were fixed and permeabilized by fixation buffer and intracellular staining perm wash buffer (Biolegend), and stained with anti-IFN-γ, anti-MIP-1α, anti- MIP-β, anti- TNF-α, and anti-GzmB as described(Yao et al., 2013 (link)). Then Boolean gates were applied to analyze (Precopio et al., 2007 (link)).
Li C., Zhu B., Son Y., Wang Z., Jiang L., Xiang M., Ye Z., Beckermann K.E., Wu Y., Jenkins J., Siska P.J., Vincent B.G., Prakash Y.S., Peikert T., Edelson B.T., Taneja R., Kaplan M.H., Rathmell J.C., Dong H., Hitosugi T, & Sun J. (2019). The transcription factor Bhlhe40 programs mitochondrial regulation of resident CD8+ T cell fitness and functionality. Immunity, 51(3), 491-507.e7.
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6

MHC-I Antigen Presentation in Macrophages and DCs

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Immature BMDCs as well as the B6 macrophage cell line were incubated in the dark ±0.2 µg/ml TPCS2a overnight in 24-well plates (1 × 105). TPCS2a was washed away from the cells (PBS, 3×) and the cells were incubated for 4 h in the presence of peptide antigens as indicated. OVA257–264 peptide (SIINFEKL), N-terminally extended OVA248–264 (9 + SIINFEKL), and C-terminally extended OVA257–280 (SIINFEKL + 15) were used for stimulation (all from Anaspec). Cells were illuminated for the indicated periods and incubated overnight in the dark. Cells were stained with anti-CD11c FITC (clone HL3, BMDCs) or anti-CD11b FITC (clone M1/70, B6 macrophage cell line) and anti-H-2Kb-SIINFEKL PE (clone 25-D1.16, eBioscience), specifically recognizing OVA257–264 (SIINFEKL) peptide bound to MHC class I (H-2Kb). The anti-H-2Kb-SIINFEKL signal was quantified on CD11c+ cells for CD11b+ cells for the B6 cell line, respectively. Cells were analyzed on a BD LSRII flow cytometer and data analyzed using FlowJo and GraphPad Prism software.
Haug M., Brede G., Håkerud M., Nedberg A.G., Gederaas O.A., Flo T.H., Edwards V.T., Selbo P.K., Høgset A, & Halaas Ø. (2018). Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination. Frontiers in Immunology, 9, 650.
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7

Murine DC Generation and Antigen Pulsing

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The development of DCs from murine bone marrow (BM) progenitor cells was performed as previously published (21 (link)). BM cells were cultured overnight in RPMI 1640 (Life Technologies) with 10% FCS, 1% penicillin, streptomycin and amphotericin in a Petri dish. Nonadherent cells were replated on day 1 at 1 x 105 cells/well in 6-well plates with murine interleukin-4 (IL-4 500 U/mL; R&D Systems) and murine granulocyte-macrophage colony stimulating factor (GM-CSF 100 ng/ml; Amgen) for 7 days. DC were resuspended at 2–5×106 cells/ml in serum-free RPMI and pulsed with OVA257–264 peptide (AnaSpec) at a concentration of 10μM in serum-free media for 90 min at room temperature.
Tavaré R., Escuin-Ordinas H., Mok S., McCracken M.N., Zettlitz K.A., Salazar F.B., Witte O.N., Ribas A, & Wu A.M. (2015). An effective immuno-PET imaging method to monitor CD8-dependent responses to immunotherapy. Cancer research, 76(1), 73-82.
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8

T Cell Stimulation and Signaling Assays

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For T cell stimulations anti-CD3, clone 145-2C11 (BD 553057) and anti-CD28, clone 37.51 (BD 553295) were used. For immunoprecipitations (IP) and immunoblotting (IB): Mouse anti-Dlg1 (BD 610875), Rabbit anti-p38, clone C20 (Santa Cruz Biotechnology sc-535), Mouse anti-Lck, clone 3A5 (Santa Cruz Biotechnology sc-433), Mouse anti-ZAP70 (BD 610239) Mouse anti-WASp, clone B-9 (Santa Cruz Biotechnology, sc-13139), Rabbit anti-phospho-p38 (T180/Y182), clone D3F9 (Cell Signaling 4511), Donkey Anti-Mouse IgG-HRP (Jackson ImmunoResearch 715-035-150), and Donkey-Anti-Rabbit IgG-HRP (Santa Cruz Biotechnology sc-2305) were used. For flow cytometry, Rat anti-CD8b-PE, clone H35-17.2 (BD 550798), Alexa Fluor647 Mouse anti-p38 MAPK (pT180/pY182), clone 36 (BD 612595), Rat anti-IFNγ-APC, clone XMG1.2 (BD 554413), Rat anti-IL-2-APC, clone JES6-5H4 (BD 554429), Rat anti-TNFα-APC (BD 554420), Rat anti-CD107a-APC, clone 1D4B (BD 560646), Alexa Fluor647 Phalloidin (Molecular Probes, Invitrogen A22287), Mouse monoclonal anti-WASP 26E6 [22 (link)] and Alexa Fluor647 AffiniPure F(ab)2 Donkey Anti-Mouse IgG (Jackson ImmunoResearch 715-606-150) were used. For inhibitors studies Insolution SB203580 (Calbiochem 559398), cytochalasin D (Sigma C8273) and DMSO (Sigma D2650) were used. For antigen experiments OVA257-264 peptide from AnaSpec (#60193) was used.
Silva O., Crocetti J., Humphries L.A., Burkhardt J.K, & Miceli M.C. (2015). Discs Large Homolog 1 Splice Variants Regulate p38 –Dependent and –Independent Effector Functions in CD8+ T Cells. PLoS ONE, 10(7), e0133353.
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9

Naive CD8+ T Cell Activation and Cytokine Profiling

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Naïve CD8+ T cells were purified from splenocytes of OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) RGMb flox/flox and OT-I RGMb flox/flox CD4-Cre+ mice using the Naïve CD8+ T cell isolation kit (Miltenyi Biotec, Cat#130-093-543). The purified naïve CD8+ T cells were labeled with 5μM Cell Trace Violet Proliferation dye (Thermo Fisher Scientific Cat#C34557), and co-cultured with BMDCs at the ratio of 5:1 in the presence of 100U/ml of IL-2 (PeproTech Cat#200-02) and OVA257-264 peptide (Anaspec Cat#AS-60193-1) for 72hrs. For measurement of cytokine production, the cells were restimulated with Golgi inhibitors and PMA/Ionomycin for 5 hrs, followed by intracellular cytokine staining.
Takayama H., LaRochelle W.J., Sharp R., Otsuka T., Kriebel P., Anver M., Aaronson S.A, & Merlino G. (1997). Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proceedings of the National Academy of Sciences of the United States of America, 94(2), 701-706.
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10

Isolation and Activation of Cytotoxic T Cells

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T cells were isolated from C57BL/6J or CD4.cre.tg Rosa26.LSL.tdTomato.cki OT-I.TCR.tg (OT-I−/− and OT-I+/+) mice. Spleens were collected and dissociated with the end of a plunger from a 1 ml syringe into 10 ml of PBS before filtration through a 70 μm cell strainer. T cells were isolated for intravenous injection using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies). A total of 4 × 106 isolated T cells was injected intravenously per mouse.
To generate cytotoxic T cells from OT-I mice, splenocytes were isolated and stimulated with 10 nM OVA257-264 peptide (AnaSpec) in complete medium containing Gibco RPMI 1640 (Thermo Fisher Scientific) with 10% Gibco fetal calf serum (Thermo Fisher Scientific), 2 mM l-glutamine, 50 IU ml−1 Gibco penicillin–streptomycin (Thermo Fisher Scientific) and 50 μM β-mercaptoethanol. After 3 days of stimulation, cells were resuspended in complete medium with 10 IU ml−1 recombinant human IL-2 (rHIL-2). Cytotoxic T cells were kept at a density of 0.5 × 106 cells per ml and fresh complete medium with rHIL-2 was added every 48 h. Cytotoxic T cells were used between 6 and 8 days after primary in vitro stimulation.
Ortiz-Muñoz G., Brown M., Carbone C.B., Pechuan-Jorge X., Rouilly V., Lindberg H., Ritter A.T., Raghupathi G., Sun Q., Nicotra T., Mantri S.R., Yang A., Doerr J., Nagarkar D., Darmanis S., Haley B., Mariathasan S., Wang Y., Gomez-Roca C., de Andrea C.E., Spigel D., Wu T., Delamarre L., Schöneberg J., Modrusan Z., Price R., Turley S.J., Mellman I, & Moussion C. (2023). In situ tumour arrays reveal early environmental control of cancer immunity. Nature, 618(7966), 827-833.
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