Cell culture insert falcon membrane

Trans-well co-cultures were carried out in 12-well plates using Cell Culture Insert Falcon Membrane (25 mm diameter, 0.4 μm pore size, Becton–Dickinson). For preparation of exosome-donor cells, CD4⁺ T lymphocytes were activated for 48 h, and then infected with spinoculation with 200 ng CAp24 equivalents of (VSV-G) wtHIV-1/10⁶ cells or equivalent amounts of the Δnef derivative. After additional 72 h, the percentage of HIV-1 infected cells was evaluated by Gag-specific FACS analysis. In the presence of at least 25 % of infected cells, cell cultures were seeded in the upper chamber of trans-well plates while HIV-1 latently infected autologous CD4⁺ T lymphocytes obtained as described above were seeded in the bottom chamber. AZT was then added to the co-cultures which were run for 48 h in the presence or not of 1 μM of either GW4869 (Sigma) and spiroepoxide (Santa Cruz) inhibitors of exosome synthesis [39 (link)–43 (link)], TAPI-2 [30 (link)], or anti-TNFα Abs. Thereafter, target lymphocytes seeded in the bottom chambers were analyzed by FACS for the expression of intracytoplasmic HIV-1 CAp24.

Trans-well co-cultures were carried out in 12-well plates using Cell Culture Insert Falcon Membrane (25 mm diameter, 0.4 μm pore size, Becton Dickinson). They were set up by putting 10⁶ F12/Hut-78 or parental Hut-78 cells in the upper chamber, while 2 × 10⁶ unstimulated CD4⁺ T lymphocytes were seeded in the bottom chamber. The co-cultures were then run overnight in the presence or not of 1 μM of the inhibitors of exosome release GW4869 (Sigma) and Spiroepoxide (Santa Cruz). Then, CD4⁺ T lymphocytes were recovered and infected with HIV-1, washed, and left in culture 3 days in the presence or not of 10 μM AZT. Afterwards, lymphocytes were analyzed by FACS for the HIV-1 expression through the detection of intracytoplasmic HIV-1 CAp24.