Computational protocol: HIV-1 Nef Impairs Key Functional Activities in Human Macrophages through CD36 Downregulation

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Protocol publication

[…] For each sample, 1×105 cells were suspended in Ca2+Mg2+-free Phosphate Buffered Saline (PBS), supplemented with 0.5% BSA, and labeled with the following anti-human antibodies: AlloPhycoCyanin (APC)-H7-conjugated CD14, Fluorescein IsoThioCyanate (FITC)- or APC-conjugated CD36 (anti-thrombospondin receptor), phycoerythrin (PE)-conjugated CD86, PE-conjugated CD206, APC-conjugated CD68, FITC-conjugated CD11c (all from BD Biosciences, Erembodegem, Belgium), PE-conjugated Toll Like Receptor-2 and 4 (TLR-2 and TLR-4, Serotec, Düsseldorf, Germany), or appropriate isotype controls. All the antibodies were incubated at the concentration of 1 μg/106 cells for 30 min in the dark on ice unless otherwise advised by manufacturers. Dead cells were excluded by Sytox Blue staining (1 μM, Molecular Probes, Carlsband, CA, USA). Intracytoplasmic staining of CD68 was performed by using BD Cytofix/Cytoperm Kit (BD Biosciences) and dead cells were excluded from the analyses by Fixable Viability Dye eFluor 780 staining (eBioscience, San Diego, CA, USA). For lymphocyte and MDM purification, cells were isolated from the culture bulk by cell sorting on the basis of their forward scatter. The purity of sorted population was found >95% after reanalysis.Stained cells were analyzed or sorted by using a BD FACSAria (BD Biosciences), equipped with three lasers (488 nm, 635 nm and 407 nm), and the results were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences) or FlowJo Software version 7.6.1 (Tree Star, Inc., Ashland, OR, USA). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases Keratoderma, Palmoplantar, Opportunistic Infections, Macrophage Activation Syndrome