Materials and Reagents

1. 15 ml polypropylene conical tubes (Corning, catalog number: 430790 )
   
2. 50 ml polypropylene conical tubes (Corning, catalog number: 430828 )
   
3. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
   
4. 6-8 weeks old C57BL/6J WT mice used for donors (males)
   
5. 6-8 weeks old Immunodeficient RAG1^−/− mice (males) (The Jackson Laboratory, Stock number: 00 2216 )
   
6. 10 cm petri dish (Corning, catalog number: 430167 )
   
7. 10 ml syringe (BD Biosciences, catalog number: 301604 )
   
8. 27 gauge needle (BD Biosciences, catalog number: 305109 )
   
9. 40 μm nylon cell strainer (Corning, Falcon^®, catalog number: 352340 )
   
10. 70 μm cell filter (Corning, Falcon^®, catalog number: 352350 )
    
11. Polystyrene round-bottom flow tubes (Corning, Falcon^®, catalog number: 352054 )
    
12. FBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 10100-147 )
    
13. RPMI media 1640 (Thermo Fisher Scientific, Gibco^TM, catalog number: 11875-093 )
    
14. ACK lysing buffer for erythrocyte lysis (Thermo Fisher Scientific, Gibco^TM, catalog number: A10492-1 )
    
15. Mouse CD4 Negative Isolation Kit (Miltenyi Biotec, catalog number: 130-095-248 )
    
16. Fluorescently labeled antibodies:
1. anti-CD25-PE (BioLegend, catalog number: 102012 )
   
2. anti-CD4-PE-Cy7 (BD Biosciences, catalog number: 563933 )
   
3. anti-CD44-APC (BD Biosciences, catalog number: 559250 )
   
4. anti-CD62L-FITC (BD Biosciences, catalog number: 553150 )
17. 37% Formaldehyde (formalin) Solution (Avantor Performance Materials, J.T.Baker^®, catalog number: 2016 )
    
18. FACS buffer (see Recipes)

Equipment

1. Plain glass microscope slides (Omano, catalog number: OMSK-50PL )
   
2. Hemocytometer
   
3. Light microscope
   
4. Top-loading balance
   
5. Centrifuge
   
6. Flow cytometry (BD Biosciences, model: ARIA III )
   

Procedure