Visualization of IPMCs in MDMs. (A-C) Uninfected MDMs were labeled with 5 μg/ml FM 4-64FX, fixed and analyzed by confocal microscopy. (A) Shows a single optical section, (B) a three-dimensional reconstruction assembled from 131 optical z-slices (step size of 0.04 μm), and (C) a detail of the IPMC shown in B. See also Additional file 1. (D-F) Live cell imaging of uninfected MDMs labeled with CellMask; (D) shows a single section, (E) a three-dimensional reconstruction assembled from 165 optical z-slices (step size of 0.1 μm), and (F) a detail of the IPMC. See also Additional file 3. Cells were imaged using an UltraVIEW Vox spinning disc confocal system (PerkinElmer, Cambridge, UK) fitted on a Nikon ECLIPSE Ti microscope equipped with a temperature and CO2-controllable environment chamber. (G,H) Uninfected MDMs (G) or MDMs infected with HIV-1 for 7 days (H) were labeled with the membrane-impermeable dye CellMask and fixed. The images show single confocal sections. Arrowheads mark CellMask-stained IPMCs. (I-K) Uninfected MDMs were nucleofected with an expression plasmid encoding PH-GFP and 24 hours later were fixed and imaged by confocal microscopy; (I) shows a single section, (J) a three-dimensional reconstruction assembled from 230 optical z-slices (step size of 0.04 μm), and (K) a detail of the IPMC. See also Additional file 4. The confocal images were acquired with a Leica TCS SPE confocal microscope, 63× oil objective (NA 1.3) and Leica LAS Software, and processed using Adobe Photoshop, ImageJ and Fiji. All experiments were repeated with cells from at least three different donors. All scale bars = 10 μm. IPMC: intracellular plasma membrane-connected compartments; PH-GFP: Phospholipase Cδ pleckstrin homology domain linked to GFP.
Mlcochova et al. BMC Biology 2013 11:89 doi:10.1186/1741-7007-11-89