12 mm circle cover slips

BMMs were cultured on 12 mm circle cover slips (Thermo Fisher Scientific) in 24-well plates with the above described cell density and stimulation protocol. At day 4 the cells were fixed and stained with Acti-stain 670 phalloidin (Cytoskeleton, Inc.), according to the manufacturer's instructions, to visualize the F-actin ring formation. After washing with PBS, cover slips were mounted with Fluoroshield^TM with DAPI (Sigma), and transferred upside down on object slides.

Internalization of the dendrimers into cells was analyzed by confocal microscopy with a Leica TSC SPE Confocal Microscope (Leica, Wetzalar, Germany). PBMCs were seeded at a density of 1 × 10⁶ cells/well in 24-well plates and U87MG-CD4⁺CCR5⁺ cells were seeded at 7.5 × 10³ cells in 12 mm circle cover slips (Thermo Fisher Scientific, Waltham, MA, USA) pre-treated for 24 h with poly-L-Lysine (Sigma, St Louis, MO, USA). Cells were treated with the fluorescent dendrimers for 1 h, 2 h or 6 h at 37ºC. After incubation, handling PBMCs as suspension cells and U87MG-CD4⁺CCR5⁺ as adherent cells, cells were rinsed twice with 3% bovine serum albumin (BSA, Sigma, St Louis, MO, USA) phosphate buffered saline (PBS, Lonza, Base, Switzerland), fixed with 4% paraformaldehyde (PFA, Panreac, Barcelona, Spain) and permeabilized with 0.1% Triton 100X (Sigma, St Louis, MO, USA) for 15 min. Cells were then incubated with Alexa Fluor® 555 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT for actin labelling and then rinsed with PBS 3% BSA. Lastly, cells were incubated with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, St Louis, MO, USA) for nuclear visualization and mounted in microscope slides (Dako, Carpinteria, CA, USA) with fluorescent mounting media (Dako, Carpinteria, CA, USA).