Materials and Reagents

1. Cell lines
   1. The B16.OVA melanoma cell line (Fidler, 1975; Schuler et al., 2008) maintained in cDMEM supplemented with G418-sulphate (geneticin selective antibiotic) (at 1 mg/ml)
      Note: Adherent cells detached using 0.25 % Trypsin-EDTA.
      
   
   
2. Mice
   1. 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. CD45.1 congenic (B6.SJL-PtprcaPep3b/BoyJArc), bred in-house at the SPF Animal Facility of the UNIL
      
   3. OT-I mice (Hogquist et al., 1994)
      
   4. OT-IIxFoxp3-eGFP mice [referred to as OT-II (Barnden et al., 1998, Wang et al., 2008)] bred in-house at the SPF Animal Facility of the UNIL
      
      
3. Antibodies
   1. Va2 and Vb5.1/5.2 antibodies (BD Biosciences)
      
   2. CD45 PE antibody (BD Biosciences)
      
   3. CD45.1 APC-eFluor780 (eBioscience)
      
   4. CD45.2 pacific blue (eBioscience)
      
   5. CD8 eFluor700 (eBioscience)
      
   6. CD4 PE-Texas red (eBioscience)
      
   7. 2.4G2 (Anti-FcgRII monoclonal antibody)
      
   
   
4. Buffers and media
   1. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX™ supplement (Life Technologies, Gibco^®, catalog number: 10566-016 )
      
   2. 1 M HEPES (Life Technologies, Gibco^®, catalog number: 15630-080 )
      
   3. Penicillin-streptomycin (5,000 U/ml) (Life Technologies, Gibco^®, catalog number: 15070063 )
      
   4. Fœtal bovine serum (FBS). (performance sera with low endotoxin : qualified, US origin) (Life Technologies, catalog number: 26140 or similar)
      
   5. Geneticin^® selective antibiotic (G418 Sulfate, 50 mg/ml) (Life Technologies, Gibco^®, catalog number: 15630-080)
      
   6. 0.25% Trypsin-EDTA  (1x) (phenol red) (Life Technologies, Invitrogen^TM, catalog number: 25200056 )
      
   7. Phosphate buffered saline (PBS) (Laboratorium Dr. Bichsel AG)
      
   8. Collagenase, Type I (Life Technologies, Gibco^®, catalog number: 17018-029 )
      
   9. DNAse I  (Roche Diagnostics, catalog numner: 0 4536282001 )
      
   10. UltraPure™ 0.5 M EDTA (pH 8.0) (Life Technologies, Invitrogen^TM, catalog number: 15575-020 )
       
   11. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
       
   12. Complete Dulbecco’s modified Eagle’s medium (cDMEM) (see Recipes)
       
   13. Tumor digesting buffer (see Recipes)
       
   14. Flow Buffer (see Recipes)
       
   15. MACS Buffer (see Recipes)
       
   
   
5. Peptides
   1. OVA257-264 and OVA323-339 peptides
      Note: Peptides were manufactured by the Protein and Peptide Chemistry Facility (PPCF) of the UNIL.
      
   
   
6. Adjuvants
   1. Poly (I: C) HMW  (tlrl-pic) and  Imiquimod R837 (tlrl-imq, InvivoGen)
      
   2. CpG-ODN 1826 (Coley Pharmaceuticals. No longer available. CpG-ODN 1826, tlrl-1826 from InvivoGen can be substituted.)
      
   3. Quil A saponin mix from Quillaja saponaria (Brenntag Nordic A/S)
      
   
   
7. Commercial reagents
   1. CD45 MACS microbeads, mouse (Miltenyi Biotec, catalog number: 130-052-301 )
      Datasheet available online: https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001245.ashx
      
   2. LIVE/DEAD Aqua cell stain (Life Technologies, Invitrogen^TM, catalog number: L34957 )
      
   
   

Equipment

1. Falcon™ 40 µm cell strainer (blue) (Corning, catalog number: 352340 )
   
2. 1ml BD Tuberculin Syringe & 26 g or 27 g x 0.5" BD™ PrecisionGlide Needle (BD, catalog number: 3052111 or 305109 )
   
3. Falcon™ 50 ml conical centrifuge tubes (Corning, catalog number: 352070 )
   
4. Falcon™ 15 ml conical centrifuge tubes (Corning, catalog number: 352099 )
   
5. Falcon™ 6 well, non-treated, flat-bottom tissue culture (Corning, catalog number: 351147 )
   
6. Dissection scissors or scalpel blade and fine-tipped forceps or tweezers
   
7. 10 ml BD™ syringe (catalog number: 309604 ) with 18 g or 20 g x 1" BD™ PrecisionGlide needle (BD, catalog number: 305195 or 305175 )
   
8. Haemocytometer
   
9. AutoMACS automatic cell separator (Miltenyi Biotech)
   
10. FACSCanto flow cytometers (BD)
    

Software

1. FlowJo software for Mac, version 9 software (TreeStar)
   
2. Prism Graphpad software
   Note: It was used to perform One-way Anova test combined with the Dunnet’s post test for statistical analysis of 3 samples per group, per experiment. Experiments were repeated twice for statistical robustness.
   

Procedure