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

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

  2. 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

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

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

  3. Wrap plastic foil around the plates

  4. Incubate at 37°C/5% CO2

 

Continuation of culture

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

  2. 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

  1. 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.

  2. 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

  3. 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

    1. 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.

    2. 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

      cloning.gif

Department of Microbiology
School of Medicine
University of Washington
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