Materials and Reagents

1. Tonsillar tissue obtained from routine tonsillectomy (Age range of patients: 2-10)
   
2. Cervico-vaginal tissue obtained from routine hysterectomy (Age range of patients: 35-55)
   
3. Gelfoam 12-7 mm adsorbable gelatin sponge (Pfizer, NDC: 0009-0315-08 )
   
4. RPMI 1640 (Life Technologies, Gibco^®, catalog number: 31870-025 )
   
5. 100x Modified Eagle's medium (MEM)-nonessential amino acids (10 mM) (Life Technologies, Gibco^®, catalog number: 11140-035 )
   
6. 100x MEM sodium-pyruvate (100 mM) (Life Technologies, Gibco^®, catalog number: 11360-070 )
   
7. 1,000x Gentamicin (50 mg/ml) (Corning, cellgro^®, catalog number: 30-005-CR )
   
8. 100x Fungizone (250 μg/ml, amphotericin B) (Life Technologies, Gibco^®, catalog number: 15290-018 )
   
9. Fetal bovine serum (FBS) (Gemini Bio-products, catalog number: 100-106 )
   Note: We advise testing several lots of serum for culture optimization and use the same lot of FBS for an entire series of experiments. We always test several serum lots on tissues from several donors and select the lot that gives the highest HIV-1 replication. Also, FBS can affect the ability of tissue to secrete cytokines in culture medium.
   
10. Phosphate Buffered Saline (PBS) (pH 7.4) (Life Technologies, Gibco^®, catalog number: 10010-023 )
    
11. Sterile water, cell culture grade (Quality Biological, catalog number: 118-162-101 )
    
12. HIV-1 viral preparation(s)
    Note: For most of our experiments we use the following viral preparations: HIV-1_BaL and HIV-1_LAI.04 obtained from the Virology Quality Assurance Laboratory at Rush University (Chicago, IL). Viral stocks were obtained from the clarified culture medium of peripheral blood mononuclear cell cultures inoculated with either HIV-1_BaL or HIV-1_LAI.04, originally received from the NIH AIDS Reagent Program. HIV-1 p24_gag concentrations were 49 ± 3 ng/ml and 53 ± 3 ng/ml for HIV-1_BaL and HIV-1_LAI.04 stock, respectively. For more viral preparations used in our experimental setting see Introini et al., 2013 and Vanpouille et al., 2012.
    
13. Timentin (GlaxoSmithKline, NDC: 0029-6571-26 ) (see Recipes)
    Note: Timentin is the commercial name of a mix of the antibiotics ticarcillin and clavulanate that are commercially available as individual reagents. These antibiotics efficiently prevent growth of bacteria that can occasionally contaminate tissue samples after surgery. Penicillin and streptomycin can be used instead of Timentin, although they have different properties. For example, Timentin displays low stability at room temperature or 37 °C (about 24 h) therefore, once added to culture medium, it remains active only for the first day of culture.
    
14. Culture medium (CM) (see Recipes)

Equipment

1. Petri dish (100 mm x 20 mm) (BD, Falcon^®, catalog number: 353003 )
   
2. Petri dish (150 mm x 25 mm) (BD, Falcon^®, catalog number: 353025 )
   
3. 6-well plates (Corning, Costar^®, catalog number: 3506 )
   
4. 12-well plates (Corning, Costar^®, catalog number: 3513 )
   
5. 5, 10-ml pipettes (BD, Falcon^®)
   
6. Sealed screw-cap 1.5/2-ml tubes (SARSTEDT AG)
   
7. Thermomixer with block for 1.5 ml-tubes (Eppendorf)
   
8. Forceps or tweezers
   
9. Scalpels and blades nos. 10 and 23
   
10. Scissors
    
11. Flat weighing metallic spatula
    
12. 37 °C, 5% CO₂ incubator set at 90% humidity
    
13. Water bath
    
14. 10, 50-ml syringe plunger
    

Procedure