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).
Materials and Reagents
Equipment
Software
Procedure
Note: While doing the staining of PBMCs with anti CD14 magnetic beads, always do the staining in the fridge rather than on ice in order to prevent antibody ‘capping effect’.
Data analysis
Notes
Recipes
Acknowledgments
We thank Alice F. Liang, Michael Cammer and the NYULMC OCS Microscopy Core for the service provided for light microscopy; Wendy Lin for technical help; and Jarrod Johnson, Nicolas Manel, for their critical advice. This work was supported by fellowships from EMBO, the Cancer Research Institute, and the Philippe Foundation (M.M.M.); by the Howard Hughes Medical Institute (D.R.L.) and Helen and Martin Kimmel Center for Biology and Medicine (D.R.L.); and by grants from the National Institutes of Health (R21AI084633) (D.R.L.) and NCRR (S10RR023704-01A1).
References
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