Cytometry.

To assess HIV-infected lymphocytes in human tissues ex vivo, we isolated tonsillar cells by treating them with Liberase DL as previously described (19) and stained them with a LIVE/DEAD dye (Invitrogen) and a combination of the following fluorescence-labeled monoclonal antibodies: anti-human CD3-Pacific Blue (PB), anti-human CD4-QD605, anti-human CD8-QD705, anti-human CD45-allophycocyanin (APC) (Caltag Laboratories, Burlingame, CA), and anti-p24-phycoerythrin (PE) (Beckman Coulter, Brea, CA). Cells were acquired on a BD LSRII flow cytometer equipped with 355-, 407-, 488-, 532-, and 638-nm laser lines (BD Biosciences, San Jose, CA).

To assess cellular proteins incorporated into HIV, virions produced by tonsillar tissue exposed to irradiated monocytes with adsorbed virus or by purified macrophages were captured with anti-2G12 magnetic nanoparticles (MNPs), as previously described (20). Briefly, 15-nm iron oxide MNPs (Ocean NanoTech, Springdale, AR) were coupled with 1 mg of 2G12 antibodies (Polymun Scientific, Austria) according to the manufacturer's protocol. To capture viruses, labeled 2G12-MNPs were incubated with HIV preparations at 37°C for 40 min, and then, complexes with captured virus (HIV-2G12-MNPs) were stained either with anti-CD36-PE antibodies (BioLegend, San Diego, CA) or with the isotype control antibody mouse IgG2a-PE (BioLegend). The resultant complexes were separated from unbound fluorescent antibodies on a magnetic column (Miltenyi Biotech, Germany) in a strong magnetic field generated by an OctoMacs magnet (Miltenyi Biotech) and washed three times with 500 μl of washing buffer (0.5% bovine serum albumin, 2 mM EDTA in PBS). To elute the captured and stained viruses, the column was removed from the magnet and demagnetized, and the complexes were eluted in 400 μl of PBS and fixed with a solution of 1% paraformaldehyde (PFA). The eluted complexes were analyzed on the LSRII cytometer triggering on fluorescence.