Materials and Reagents

1. Tissue specimens of small (terminal ileum) and large intestine
   
2. RPMI 1640 (Life Technologies, catalog number: 21875-034 )
   
3. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F7424 )
   
4. HEPES (Life Technologies, Gibco^®, catalog number: 15630-080 )
   
5. Penicillin and Streptomycin (Life Technologies, catalog number: 15140-122 )
   
6. HBSS (Life Technologies, catalog number: 14180-046 )
   
7. EDTA (Life Technologies, catalog number: AM9261 )
   
8. Liberase TM (Roche Diagnostics, catalog number: 05401127001 )
   
9. DNase I (Sigma-Aldrich, catalog number: D4263 )
   
10. Collagenase 1A (0.2 μm-filtered) (Sigma-Aldrich, catalog number: C9891 )
    
11. FACS antibodies
    1. T cells
       CD3-PE-Cy7 (SK7) (BD biosciences)
       Pacific blue (PB)-CD8 (RPA-T8) (BD biosciences)
       Quantum dot (QD) 605-CD4 (S3.5) (Life Technologies, Invitrogen^TM)
       LIVE/DEAD^® Fixable Near IR Dead Cell Stain Kit (Life Technologies, Invitrogen^TM)
       
    2. DC/macrophages
       CD3-PE-Cy5 (UCHT1) (eBioscience)
       CD19-PE-Cy5 (HIB19) (eBioscience)
       CD11c-PE-Cy7 (3.9) (eBioscience)
       CD103-PE (Ber-ACT8) or CD103-eFluor647 (B-Ly7) (eBioscience)
       CD14-eFluor450 (61D3) (eBioscience)
       HLA-DR-APCeFluor780 (L43) (eBioscience)
       CD20 PE-Cy5 (2H7) (BioLegend)
       TCRab-PE-Cy5 (IP26) (BioLegend)
       Biotin-or PE-Cy7 CD172a (SE5A5) (BioLegend)
       CD56-PE-Cy5 (Alpha Diagnostic Intl)
       Biotin- or FITC-CD11c (MJ4-27G12) (Miltenyi Biotec)
       CD141-PE (AD5-14H12) (Miltenyi Biotec)
       CD45 V500 (HI30) (BD Biosciences)
       Biotinylated antibodies were detected using streptavidin conjugated to PE-Cy7 (eBiosicence) or QDot605 (Life Technologies, Invitrogen^TM).
       Dead cells were excluded from analysis using propidium iodide PI (Life Technologies, Molecular Probes^®).
       
12. R10 medium (see Recipes)
    

Equipment

1. Cell strainer (100 μm) (Thermo Fisher Scientific, catalog number: 22363549 )
   
2. 50 ml Falcon tube
   
3. Polyester filters cut in 10 x 10 cm squares (mesh count 27 threads/cm, mesh opening 250 μm, thread diameter 120 μm) (Tekniska Precisionsfilter JR AB)
   
4. Petri dish (SARSTEDT AG, catalog number: 82.1473 )
   
5. Ultra low attachment 24-well culture plate (Sigma-Aldrich, catalog number: CLS3473 )
   
6. Pipette
   
7. Needle/forceps
   
8. Surgical scissor/scalpel
   
9. Beakers with lid (VWR International, catalog number: 216-2694 )
   
10. 37 °C, 5% CO₂ cell culture incubator
    
11. Shaker
    
12. Centrifuge
    
13. Microscope
    
14. Multicolour FACS analyser (BD Lsr II flow cytometer)
    

Software

1. FlowJo software (Tree Star Inc)

Procedure