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Figure 2 | Retrovirology

Figure 2

From: Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

Figure 2

DTNB inhibits fusion/entry of BlaM-containing virions in JC53 cells, primary human PBMCs and MDM. The BlaM-containing virus particles were produced and the JC53 CD4+/CCR5+ cells, primary human MDM, and PBMCs were cultured as described in Methods. DTNB was dissolved in pre-warmed phenol red free DMEM, and cells were pre-incubated for 1 h before addition of the virus. The infection period was 2 h at 37°C in serum-free medium for the JC53 cells (panel A) and 2.5 h for the primary PBMCs (panel B) and MDM (panel C) in the presence of DTNB. After incubation with BlaM-containing virus particles, cells were washed with DMEM, loaded with the fluorescent dye CCF2/AM for 1 h at RT, washed and further incubated in DM-10 (phenol red free, supplemented with 2.5 mM Probenecid) for 14 h in tissue culture plates at RT in the dark. Subsequently, the JC53 cells (after trypsinization) and the human PBMCs were transferred to microfuge tubes, fixed with 1.6% pareformaldehyde and analyzed using BD LSR II Cell Fluorometer equipped with a violet laser (407 nm excitation wavelength) and 460/30 nm and 530/20 nm emission filters for detection of the blue and green fluorescence, respectively (A,B). The primary human MDM (C), cultured in a borosilicate glass bottom plate (Whatmann), were fixed in situ and analyzed using a Laser Scanning Cytometer (CompuCyte, Cambridge, MA). Mock-infected cells (upper panels) were used to define the regions of negative (green) and positive (blue) cell populations in the presented histograms. The experiment with human MDM was performed in triplicates. The infection of the JC53 cells and primary PBMC was carried out in duplicate wells, which were combined before Flow Cytometry analysis.

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