Ex vivo Co-culture of Lymphoid Tissue Stromal Cells and T Cells

Materials and Reagents

1. RPMI-1640 medium (Life Technologies, catalog number: 11875-093 )
   
2. Fetal bovine serum (FBS) (GEMBIO, catalog number: 900-108 )
   
3. Antibiotic-Antimycotic (Life Technologies, catalog number: 15240-062 )
   
4. Anti-CD45RA (Dako, catalog number: M0754 )
   
5. Anti-activated caspase-3 (Cell Signaling Technology, catalog number: 9665 )
   
6. Anti-CD3 (AbD Serotec, catalog number: MCA1477 )
   
7. Anti-IL-7 (R&D Systems, catalog number: MAB207 )
   
8. Streck's tissue fixative (Streck Laboratories, catalog number: 265138 )
   
9. TritonX-100 (Sigma-Aldrich, catalog number: X-100 )
   
10. CellMicroSieves ( BioDesign Inc. of New York, catalog number: N100S )
    
11. Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies, Invitrogen^TM, catalog number: A-21206 )
    
12. Alexa Fluor 568 Donkey Anti-Mouse IgG (Life Technologies, Invitrogen^TM, catalog number: A10037 )
    
13. Alexa Fluor 647 Chicken Anti-Rat IgG (Life Technologies, Invitrogen^TM, catalog number: A-21472 )
    
14. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    
15. Phosphate-BufferedSaline(PBS)(LifeTechnologies,Invitrogen^TM,catalog number: 10010-023 )
    
16. Complete RPMI-1640 culture medium (see Recipes)
    Note: The experimental protocols used here for human tissue samples had full IRB approval (Institutional Review Board: Human Subjects Committee, Research Subjects’ Protection Program, University of Minnesota) and informed written consent was obtained from individual patients, or the legal guardians of minors, for the use of tissue in research applications prior to the initiation of surgery.
    

Equipment

1. Biological Safety Cabinet
   
2. Chamber slides (Thermo Fisher Scientific, catalog number: 154526 )
   
3. Centrifuges
   
4. Water bath
   

Procedure