To begin, seed 0.1 x 10 to the power of six TZM-bl cells in a 24-well plate using 500 microliters of complete DMEM for each well, and place it in a cell culture incubator for 10 to 12 hours. From the vial containing the HIV-1 virus, transfer an appropriate amount of the virus to the wells and incubate at 37 degrees Celsius for four hours. Wash the cells twice with one milliliter of PBS before fixing them in 500 microliters of 0.25%glutaraldehyde solution.
Incubate the cells at room temperature for five to seven minutes. After the PBS wash, overlay the cells with 500 microliters of freshly prepared staining solution and incubate at 37 degrees Celsius in the dark for two to 18 hours. Under the microscope at 10X magnification, count the blue infected cells for five random fields.
Resuspend the 8 million CEM-GFP cells in one milliliter of polybrene containing complete RPMI 1640 medium. Add the virus stock and make up the volume to two milliliters with complete medium. Place the cells in a 37 degrees Celsius carbon dioxide incubator for four hours, with intermittent tapping every 30 to 45 minutes.
Culture an equal number of cells in a six-well culture plate at 37 degrees Celsius. Every 24 hours, after centrifugation, collect the supernatant from pelleted cells and add cell lysis buffer into the pelleted cells.
