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4 protocols using ova257 264 siinfekl peptide

1

Quantifying SIINFEKL-Presenting DCs in dLN

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To analyze OVA257–264 (SIINFEKL) peptide (Anaspec) presenting DCs, dLNs were isolated and digested on day 7 post vaccination. dLN cells were enumerated and stained with APC conjugated anti-mouse CD11c (eBioscience 17–0114) and PE conjugated anti-mouse SIINFEKL peptide bound to H-2Kb (eBioscience 12–5743) for 15 minutes on ice. Cells were washed and assessed by flow cytometry.
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2

Quantifying SIINFEKL-Presenting DCs in dLN

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To analyze OVA257–264 (SIINFEKL) peptide (Anaspec) presenting DCs, dLNs were isolated and digested on day 7 post vaccination. dLN cells were enumerated and stained with APC conjugated anti-mouse CD11c (eBioscience 17–0114) and PE conjugated anti-mouse SIINFEKL peptide bound to H-2Kb (eBioscience 12–5743) for 15 minutes on ice. Cells were washed and assessed by flow cytometry.
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3

Immunomodulation Mechanism Analysis

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p38 MAPK inhibitors SB202190 and SB203580 (Sigma-Aldrich, St. Louis, MO, USA), PP2A inhibitors Okadaic acid (Sodium Salt, EMD Millipore, Billerica, MA, USA) and Endothall (Sigma-Aldrich), DUSP1/6 Inhibitor (BCI, EMD Millipore); OVA (257–264)/SIINFEKL peptide (Anaspec, Fremont, CA, USA), recombinant endotoxin-free OVA protein (Invivogen, San Diego, CA, USA), Toll-like receptor (TLR) agonists LPS (O127:B8 E. coli strain, Sigma-Aldrich), and mouse IL-6 ELISA and mouse IL-1β ELISA sets (BD Pharmingen, San Diego, CA, USA). Other antibodies and protein standards were purchased from Biolegend including purified antimouse IL-12 (p70) antibody (C18.2), biotin antimouse IL-12/IL-23 p40 antibody (C17.8), recombinant mouse IL-12 (p70) (ELISA Std.), purified antimouse IFN-γ antibody (AN-18), biotin antimouse IFN-γ antibody (R4-6A2), recombinant mouse IFN-γ (ELISA Std.), purified antimouse IL-4 antibody (11B11), and biotin antimouse IL-4 antibody (BVD6-24G2).
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4

Mouse BMDC Maturation and Antigen Priming

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Murine GM-CSF and human Flt3-L (both from Peprotech, Rocky Hill, NJ) were used to derive BMDCs from mouse bone marrow. CpG-Oligodeoxynucleotide (ODN), polyinosinic-polycytidylic acid [Poly(I:C)], monophosphoryl lipid A (MPLA) (all from Invivogen, San Diego, CA), and lipopolysaccharides (LPS) (Sigma, St Louis, MO) were used as adjuvants to induce maturation of BMDCs. Ovalbumin (OVA, model protein antigen unless specified otherwise) (EndoFit, Invivogen), fluorescently (Alexa Fluor 555)-labeled OVA (Molecular Probes, Eugene, OR), or OVA (257–264) SIINFEKL peptide (AnaSpec, Fremont, CA) was used as a model antigen. Ultra-pure chitosan (MW <200 kDa, deacetylation > 75%, NovaMatrix, Sandvika, Norway) was used to prime the mouse vaginal tract.
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