HIV uses chemokine receptor like CCR5 and/or CXCR4 as the major coreceptor along with CD4 receptor to gain entry into the cell. Coreceptor usage has also been shown to correlate disease progression and SI phenotype of the virus. In laboratory, coreceptor usage of an isolate can be determined by using GHOST cells - Human osteosarcoma cells transfected with genes coding for CD4 receptor, CXCR4/CCR5 coreceptors and the green fluorescent protein (GFP) as the reporter gene which is under the control of HIV-2 tat. Upon infection, the GFP is transactivated by tat, leading to the production of GFP in the cytoplasm. Infected cells emit fluorescence, which can be measured cytofluorometrically.  

Procedure:

1. Seed 24-well plates with 70 x103 GHOST-CXCR4/CCR5 cells per well resuspended in 0.5ml 10%DMEM                         
2. Incubate the plates at 37oC in presence of 5%CO2 for 24 hours.
3. Remove the medium using suction without letting the cell monolayer to dry.
4. Add 75µl of virus and 75µl of 10%DMEM to each well in duplicate. Add 50µl of DEAE-Dextran (32µg/ml). Final concentration of DEAE-Dextran should be 8µg/ml.
5. Incubate the plates at 37oC in presence of 5%CO2 for 24 hours.
6. Remove the virus-medium mixture with suction and wash the cell monolayer with plain DMEM.
7. Add 1ml 10% DMEM to each well and Incubate the plates at 37oC in presence of      5%CO2 for 24 to 72 hrs. Observe plates daily for CPE and cell detachment.  If there is good CPE and more detachment, harvest cells as follows.
8. Wash the wells with plain DMEM and add 300µl of 1mM EDTA in PBS in each well to detach the cells.
9. Label the Falcon tubes. Add 150µl of 6%formaldehyde to each tube. Transfer resuspended cells to the appropriate tubes using multichannel pipette.
10. Acquire 15,000 cell events using Cell Quest Software, BD FACSort machine.
11. Analyse data using similar software.
12. Determine the no. of infected cells by using a scattergram of fluorescence Vs FSC (forward scatter) after setting a gate with uninfected cells.
13. Calculate the cut off value by using following formula:

Mean no. of fluorescent cells in the uninfected cell culture + 3SD