To determine the effect of Vpu on cell surface expression of tetherin and CD4, TZM.bl cells, expressing tetherin and CD4, were transfected with IMCs. 48 h post transfection cells were separately stained for surface tetherin (allophycocyanin (APC)-conjugated anti-human tetherin antibody from BioLegend, San Diego, CA, USA) and CD4 (APC-conjugated anti-human CD4 from eBiosciences, San Diego, CA, USA), as per the manufacturer’s instruction. Cells were analyzed by Attune flow cytometer, and >100,000 events were collected. Analysis was carried out using FCS-Express software as follows: TZM.bl cells were selected from a plot of forward-area vs. side scatter-area (FSC-A/SSC-A) from which doublets were excluded in a forward scatter height vs. forward scatter area plot (FSC-H/FSC-A). Live cells were selected by Aqua-negative gating, and geometric mean fluorescent intensity (MFI) of APC+ cells, representing anti-CD4 or anti-tetherin stained cells, were quantified. Background MFI, as determined from cells stained with APC isotype control was subtracted. Vpu-mediated tetherin or CD4 down-modulation is presented as median fluorescence intensity (MFI).
Apc conjugated anti human cd4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APC-conjugated anti-human CD4 is a flow cytometry reagent used for the identification and enumeration of CD4-positive cells. The CD4 molecule is a glycoprotein expressed on the surface of certain T lymphocytes, monocytes, and macrophages. The APC fluorochrome is used to label the anti-human CD4 antibody, allowing for the detection of CD4-positive cells in a sample by flow cytometry.
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2 protocols using apc conjugated anti human cd4
1
Vpu Modulates Tetherin and CD4 Expression
2
Th17 and Treg Cell Detection
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For detection of Th17 cells, PBMCs (2 × 106 cells/ml) were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) containing ionomycin (1 μμ) and Monensin (500 ng/ml). Upon harvesting, the cells were stained for CD4 by incubating the cells with fluorescein isothiocyanate (FITC)-conjugated antihuman CD4 (BioLegend) at 4°C for 30 min. After fixation and permeabilization, the cells were further stained with phycoerythrin (PE)-conjugated antihuman IL-17A (eBioscience, San Diego, CA, USA) for detection of Th17 cells. For detection of Treg cells, PBMCs were incubated with allophycocyanin (APC)-conjugated antihuman CD4, PE-Cy5-conjugated antihuman CD25, and FITC-conjugated antihuman CD127 (eBioscience) in the dark for 30 min at room temperature. Finally, the cells were analyzed by multicolor flow cytometry on a FACSCalibur (Becton Dickinson).
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