Reagents:

• U87 T4 co-receptor cell lines (mycoplasma free):

• CD4 only

• CD4 plus CCR1

• CD4 plus CCR2b

• CD4 plus CCR3

• CD4 plus CCR5

• CD4 plus CXCR4

• cDMEM (DMEM, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, L-Glutamine, 10mM HEPES)

• Sterile TC 1x PBS.

• 1x Trypsin.

• Geneticin (G418), 300 µg/ml final concentration.

• Puromycin, 1 µg/ml final concentration.

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

• 24-well plate with lid (flat bottom), Costar?

Passaging:

1. U87 cell stocks are cultured in T25 flasks in 7ml cDMEM with G418 for all cell lines and puromycin for all except parental U87 T4.

2. For passaging, wash monolayer with 5ml warm PBS.

3. Add 0.5ml 1X Trypsin.

4. Incubate 2 minutes at room temperature.

5. Confirm that all cells detach.

6. Add 2ml cDMEM and pipette up and down to completely resuspend all cells.

7. Use 250 µl cell suspension to continue culture. Add 7ml cDMEM and antibiotics.

8. Pellet cells not used for passaging and resuspend at appropriate concentration for further use (i.e. 1 x 10⁵/ml for co-receptor assay).

Co-receptor assay:

1. Per virus per cell line: plate out 1 x 10⁴ cells (100 µl) in cDMEM with antibiotics in 96-well plate.

2. Inoculate with at least 100 TCID₅₀ (in no more than 100 µl).

3. After 24 hours wash with warm PBS.

4. Add 200 µl fresh cDMEM with antibiotics.

5. After 3 and 7 days, take p24 sample. Store at -20°C.

6. After 7 days, score cytopathic effect.

7. Wash monolayer with warm PBS.

8. Add 50 µl 1x Trypsin.

9. Incubate 2 minutes at room temperature.

10. Transfer each well to a new well in a 24-well plate.

11. Add 2 ml cDMEM with antibiotics

12. After 10, 14, 17 and 21 days, take p24 samples. Store at -20°C.

13. After 14 and 21 days, score cytopathic effect.

14. Assay p24 production and discard cultures.

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