I have been trying to optimize the viral transduction protocol macrophage like THP-1 cells (PMA activated). I am not sure of the time period for which I should allow the virus exposure to cells? Also please suggest the no of days up to which I should wait for finally determining p24 content and intracellular HIV-1.
the time of infection that worked for me is 2 hr, followed by wash and add fresh media. There are also various reports where they infect for as minimum as 30 min to 24 hr.
Coming to detect p24 in the infected culture supernatants, which method would you use to detect p24? Is it capture ELISA based? The minimum time frame you can look at is 12hr post infection (your detection method should be highly sensitive) which is very difficult. But in my case, at 24 hr post infection I could detect p24 titers (please see my publications in cellular microbiology or scientific reports).
How do you want to detect intracellular p24? by microscopy or FACS? if so, you can detect immediately after infection to assess for the percent cells infected with virus out of total cells (see supporting information of my cellular microbiology paper).
Hope this helps
If you want to see de novo virus production, you should wait between 36 and 48h before assaying p24 production. PMA activated THP-1 cells endocytose free virus and can release it, without infection, up to 36h post-exposure (and sometimes longer than that).
If you want to make sure that it is p24 coming from a de novo production (and not just release of input), you should include a control treated with efavirenz or AZT.
With primary human macrophages, we follow p24 production at days 3, 6, 9, 12 and 15 post-infection. We see a peak of p24 production at 9 to 12 days. In CD4 T cells, we follow p24 production at 2, 4 and 6 days post-infectionn with a peak at day 4.
Detection of intracellular p24 can be achieved by intracellular flow cytometry using the KC57-FITC or KC57-RD1 antibody by Beckman Coulter. It works very well. This method of detection is interesting because it is quantitative and can be combined with other markers.
Good luck,
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