Computational protocol: The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells

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Protocol publication

[…] For microscopy experiments, cells were infected and treated with inhibitors as described above. Cells were then adhered to poly-l-lysine (Sigma-Aldrich) coated coverslips and fixed in 2% PFA for 10 min at room temperature. Cells were subsequently washed twice with PBS and permeabilized with methanol for 20 min. Cells were then washed twice with PBS and blocked in 2% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Cells were subsequently stained for 2 h with mouse anti-CD28 (Thermo Fisher Scientific) and rabbit anti-LAMP-1 (Developmental Studies Hybridoma Bank, University of Iowa) diluted at 1:200 in 0.2% BSA in PBS, washed twice with 0.2% BSA in PBS and incubated for one and a half hours with the appropriate fluorophore conjugated secondary antibodies (Alexa-Fluor-647 conjugated anti-mouse and Cy3-conjugated anti-rabbit; Jackson ImmunoResearch, West Grove, PA) at 1:400 in 0.2% BSA in PBS. Finally, cells were washed twice with PBS and mounted on coverslips with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). Cells were imaged on a Leica DMI6000 B at 63× or 100× magnification using the FITC, Cy3, Cy5 and DAPI filter settings and imaged with a Hamamatsu Photometrics Delta Evolve camera. Images were subsequently deconvolved using the Advanced Fluorescence Deconvolution application (Lecia, Wetzlar, Germany) on the Leica Application Suite software. Co-localization analysis was conducted using Mander’s Coefficent from the ImageJ plugin JACoP, as described previously []. […]

Pipeline specifications

Software tools ImageJ, JACoP
Application Microscopic phenotype analysis
Organisms Human immunodeficiency virus 1, Viruses