Reagents:

• Target cells:
  1. 3 day PHA-stimulated PBMC (or)
  2. appropriate T cell line

• Culture media:

  1. cIMDM (IMDM, 10% FBS, Pen/Strep, 5 µg/ml Polybrene, 10 U/ml rIL-2) (or)
  2. b. cRPMI (RPMI, 10% FBS, Pen/Strep, L-Glutamine)

• 96-well microtiterplate with lid (flat bottom), Nuclon, Nunc.

• Multichannel pipette, 10-300 µl Finnpipette, Labsystems.

• MT2 cells.

Protocol:

1. Make 5-fold serial dilution of virus-containing supernatant: in 96-well plate add 30 µl virus to 120 µl medium, mix well and transfer 30 µl mixture to next 120 µl medium. Change tips! Continue to a dilution appropriate for specific virus stock (CEM-LAI ~10⁷ TCID₅₀/ml, PP-BaL ~10⁵ TCID₅₀/ml).

2. In new 96-well plate: plate out 10⁵ target cells in 100µl per well as needed.

3. In quadruplicates: add 25 µl of each serial dilution to the appropriate wells*.

4. Wrap plastic foil around the 96-well plate.

5. Incubate for 7 days at 37°C, 5% CO₂.

6. For:
   1. PHA-PBMC: plate out 10⁵ fresh target cells in 100 µl per well as needed in new 96-well plate.
      
   2. T cell lines: plate out 100 µl fresh medium as needed in new 96-well plate.

7. Remove 40 µl supernatant for testing of p24 production.

8. Resuspend the rest of the culture and transfer 40 µl containing cells and sup to the fresh 96-well plate.

9. Add 25 x 10³ MT2 cells in 100 µl per well to the original plate.

10. Wrap plastic foil around plates and incubate at 37°C, % CO₂.

11. After 3 days: score syncytia in MT2 cultures.

12. After 7 days: remove 40 µl supernatant for testing of p24 production.

13. Discard all cultures. Use the Excel spreadsheet CalculatingTCID50.xls to calculate TCID₅₀/ml.

     

     

     

    For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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