Materials and Reagents

1. C57BL/6 mice from Harlan Laboratories (the Netherlands)
   Note: All mice used in this protocol were between 6 and 12 weeks of age and were sex and age matched for each individual experiment. Three mice were used per group in each experiment.
   
2. OVA257-264 and OVA323-339 peptides
   Note: Peptides were manufactured by the Protein and Peptide Chemistry Facility (PPCF) of the University of Lausanne.
   
3. Iscove's modified Dulbecco's medium (IMDM), GlutaMAX™ supplement (Life Technologies, Gibco^®, catalog number: 31980-030 )
   
4. Penicillin-Streptomycin (5,000 U/ml) (Life Technologies, Gibco^®, catalog number: 15070063 )
   
5. 2-Mercaptoethanol (2-ME, 55 mM) (Life Technologies, Gibco^®, catalog number: 21985-023 )
   
6. Fœtal bovine serum (FBS) (performance sera with low endotoxin: qualified, US origin) (Life Technologies, catalog number: 26140 or similar)
   
7. Phosphate Buffered Saline (Laboratorium Dr. Bichsel AG)
   
8. Poly (I: C) HMW (tlrl-pic) and Imiquimod R837 (tlrl-imq, InvivoGen)
   
9. CpG-ODN 1826 (Coley Pharmaceuticals. No longer available. CpG-ODN 1826, tlrl-1826 from InvivoGen can be substitued)
   
10. Quil A saponin mix from Quillaja saponaria (Brenntag Nordic A/S)
    
11. Cytokine Mouse 10-Plex Panel for Luminex platform (Life Technologies, Novex^®, catalog number: LMC0001 ) for use with the Luminex^® 100™⁄200™ and FLEXMAP 3D^® systems
    User manual available online: http://tools.lifetechnologies.com/content/sfs/manuals/LMC0001_Protocol_Rev1.pdf
    
12. Complete iscove's modified Dulbecco's medium (cIMDM) (see Recipes)
    

Equipment

1. 1 ml BD Tuberculin Syringe & 26 g or 27 g x 0.5" BD™ PrecisionGlide needle (Beckton Dickinson, catalog numbers: 3052111 or 305109 )
   
2. Falcon™ 24 well, non-treated, flat-bottom tissue culture plates (Corning, catalog number: 351147 )
   
3. ~40 μm nylon gauze sheets cut into small squares, e.g. Nylon Mesh Lab Pak, 41 Microns Square Opening (Nitex, catalog number: 7050-1220-000-14 ) or Nylon 6 Mesh Sheet, 48 microns Mesh Size (Small Parts, catalog number: B0043D1SCE )
   
4. Plunger from 1ml BD™ Tuberculin Syringe (BD, catalog number: 309602 )
   
5. Dissection scissors and fine-nosed forceps or tweezers
   
6. Falcon™ 96 well, non-treated, U-bottom tissue culture plates (Corning, catalog number: 351177 )
   
7. Luminex^® 200™ System with xPONENT^® Software (Life Technologies, Novex^®)
   

Software

1. xPONENT^® software (Life Technologies, Novex^®)
   
2. Statistical tests were performed using GraphPad Prism software
   Note: The different groups were compared using One-way ANOVA with the Dunnett multiple comparison test, comparing all groups to the peptide alone group.
   

Procedure