Quantifying HIV-1 Virus-Like Particle Formation

HeLa cells (5 × 10⁴) were seeded on cover glass in 24-well plates and transfected with 0.3 μg of pNL43 Luc E^- R⁺ or pNL43 Luc E^- R^-, either without/with 0.1 μg of pCAGGS/mRFP-TSG101 and 0.1 μg of pcDNA/YFP-Vpr plasmids, for 16 h. The cells were then fixed in 3.7% formaldehyde, permeabilized with 0.5% Triton X100, and stained with anti-p24 mouse mAb AG3.0 [33 (link)] and FITC-conjugated sheep anti-mouse Fab’ (ICN Biomedicals). TIRF images were acquired by an Eclipse TE2000-E inverted microscope (Nikon).

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Chutiwitoonchai N., Siarot L., Takeda E., Shioda T., Ueda M, & Aida Y. (2016). HIV-1 Vpr Abrogates the Effect of TSG101 Overexpression to Support Virus Release. PLoS ONE, 11(9), e0163100.

Publication 2016

Eclipse TE2000-E inverted microscope

Cells Fitc Formaldehyde Hela cells Microscope Mouse Plasmids Sheep Triton x100 Tsg101

Corresponding Organization : Osaka University

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Variable analysis

independent variables

Transfection of HeLa cells with pNL43 Luc E- R+ or pNL43 Luc E- R-
Co-transfection of HeLa cells with 0.1 μg of pCAGGS/mRFP-TSG101 and 0.1 μg of pcDNA/YFP-Vpr plasmids

dependent variables

Localization of p24 protein in HeLa cells as observed by TIRF microscopy

control variables

HeLa cell seeding density (5 × 10^4 cells)
Transfection duration (16 h)
Fixation and permeabilization of HeLa cells
Staining of HeLa cells with anti-p24 mouse mAb AG3.0 and FITC-conjugated sheep anti-mouse Fab'

controls

Positive control: Transfection with pNL43 Luc E- R+
Negative control: Transfection with pNL43 Luc E- R-

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