Materials:

Equipment

• Single channel pipette: 0.5-10 µl, 10-100 µl, 200-1000 µl, Eppendorf

• Multichannel pipette: 5-50 µl, 50-300 µl, Finnpipette, Labsystems

• Burker Bright-line Count-chamber, Labor Optik

• Adjustable waterbath (37°C)

• Rotixa/RP centrifuge, Hettich

• 37°C/5% CO₂ incubator, Brouwer Scientific

• Biosafety cabinet, Clean Air

• Sterile tray

Disposables

• Disposable pipette: 1, 2, 5, 10, 25, 50 ml, Falcon

• Pipette tips: 1-200 µl, Costar

• Propylene conical tubes: 15 ml, 50 ml, Falcon/Nunclon

• Sterile solution basins, individually wrapped, Labcor

• Microtiter plate: 96-well flat bottom with lid, Nunclon, Nunc

• Tissue culture flask: 25 cm², 75 cm², 175 cm², Nunc

Reagents

• Complete IMDM = IMDM + 10% FBS + Pen/Strep + 5 µg/ml Polybrene

• PHA, use in complete IMDM at 1 µg/ml, Murex (HA16/17)

• rIL-2, use in complete IMDM at 20 U/ml, Boehringer Mannheim

Protocol:

Prepare PHA-stimulated target cells

1. Isolate PBMC by separation over Ficoll gradient

2. Resuspend cells in complete IMDM with PHA, 3 to 5 x 10⁶/ml

3. Incubate 2 to 3 days at 37 °C/5% CO₂

4. Spin 10 minutes at 1500 rpm

5. Resuspend cell pellet in complete IMDM with rIL-2

6. 10 x 10⁶ cells in 10 ml are needed per 96-well plate (100 µl/well)

Prepare patient PBMC

7. Thaw and count cells (resuspended in complete IMDM with rIL-2)

8. Make four 2-fold dilutions of 2.5 ml using up the total amount of cells (e.g. 100,000/ml, 200,000/ml, 400,000/ml and 800,000/ml, resulting in final concentrations of 10,000, 20,000, 40,000, and 80,000 cells/well)

Start co-cultivation in 96-well plates

9. Plate out 100 µl of PHA-stimulated target cells per well

10. Add 100 µl of patient cells per well (24 wells per dilution)

11. Wrap plastic foil around the plates

12. Incubate at 37°C/5% CO₂

Continuation of culture

13. Prepare PHA-stimulated target cells 2-3 days prior as described above

14. Every seven days:

    1. Remove 65 µl of supernatant for testing of p24 production
    2. Resuspend the rest of the culture, transfer 65 µl to a fresh plate containing 135 µl complete IMDM with rIL-2 with 100,000 target cells per well
    3. Add 25,000 MT2 cells per well to the original plate
       
    4. Score syncytia in this plate after 3 days

Expand biological virus clones

15. When fewer than 33% of wells seeded with a certain concentration are positive, the viruses in one well are thought to originate from one infected cell.

16. Wells that show evidence of HIV-1 replication in that concentration (in p24-ELISA and/or MT-2 culture) can be transferred to a tissue culture flask (25 cm²) containing 5 x 10⁶ PHA-stimulated target cells in 5 ml complete IMDM with rIL-2

17. After 7 to 14 days virus containing cell-free culture supernatants can be collected and stored at -70°C for viral stocks, cells viably frozen and 10⁶ cells stored in lysis buffer for isolation of proviral DNA

 

Calculations

18. The proportion of infected cells is determined according to the formula for Poisson distribution: F=-ln(F₀), in which F₀ is the fraction of negative cultures.

19. Since CD4⁺ T cells have been shown to be the most important target for HIV-1 in the peripheral blood, virus load is expressed as tissue culture infective doses per 10⁶ CD4⁺ T cells.




Schematic overview of biological cloning experiments