Abstract
The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.
Background:
This protocol was developed in order to assess the changes of HIV-1 internalization upon disruption of actin nucleation in human monocyte derived dendritic cells. Following a shRNA screen to identify genes important for HIV-1 transfer from dendritic cells to T cells, we observed that a disruption of actin nucleation leads to a switch from actin rich dendrites to blebs, due to an excess of actomyosin contraction. As a consequence, a decrease of HIV-1 transfer and an increase of HIV-1 internalization due to bleb retraction-driven macropinocytosis were observed. We concluded that effectors of actin nucleation and stabilization were key to maintain HIV-1 on actin-rich dendrites and to limit its endocytosis, for efficient transfer to T lymphocytes (Menager and Littman, 2016).