P24 pe

Semen cells were first stained with fluorescent antibodies such as CD3-APC-Cy7 (BioLegend, USA, clone SK7), CD4-BV605(BD, USA, clone RPA-T4), CD8-Percp/Cyanine5.5 (BioLegend, USA, clone SK1), CD56-BV421 (BioLegend, USA, clone HCD56), CCR5-FITC (BioLegend, USA, clone J418F1), CXCR4-APC (BioLegend, USA, clone RG5), p24-PE (Beckman Coulter, USA, clone KC57), and then analyzed using imaging flow cytometry.
The initial test of the staining plate contained all but one staining agent and fluorescence minus one control to determine the background staining of the channel. Cells were then acquired on an Amnis ImageStream Mk II flow cytometer (Luminex) using the INSPIRE 4.1 software with lasers set to maximum values without saturation in the brightest stains. Cell files (50,000) were collected with a cell classifier applied to the brightfield channel to capture a single-cell picture. Channels were as follows: Brightfield-Channel 1, FITC-Channel 2, PE-Channel 3, Percp/Cyanine5.5-Channel 5, BV421-Channel 7, BV605-Channel 10, APC-Channel 11, and APC/Cy7-Channel 12. Excitation lasers were used with the typical intensity settings of 405 nm (80 mW), 488 nm (100 mW), 594 nm (20 mW), and 658 nm (40 mW). All cell images were captured with the 40× objective and acquired at a rate of 200∼250 images per second. Data were analyzed using the IDEAS 6.2 (Amnis/EMDmillipore) software.

To measure intracellular activation markers, cells were permeabilized for 30min with PermWash at RT (BD Pharmingen) and subsequently stained with p24-PE (Beckman Coulter) and IFN-γ-FitC, IL2-PerCp-Cy5.5, IL-4-APC, TNF-α-PE-CF594, Mip-1β-AlexaFluor700 (all from BD Bioscience) for 30min at 4°C at a pre-determined dilution. Next, these cells were washed once with PermWash and taken up in FACS buffer (PBS+ 2%FCS) after which they were analyzed using a FACS Canto II machine. For determining CCR5 surface expression, T-cells obtained from our T-cell outgrowth model were fixed in 3.7% formaldehyde before HIV-1 infection and stored for no more than 1 week at 4°C in FACS buffer. These cells were stained in FACS buffer with CCR5 PE-Cy7 (Biolegend) for 30min at RT, washed with FACS buffer and measured. Shown in histograms are cytokine producing T-cells with samples taken 5 and 7 days after HIV-1 infection with the optimal time point being determined by the percentage of live cells (>50%) and level of HIV-1 infection, which varied per donor and per virus. Gating strategies and flow cytometric controls are represented (S3A Fig and S3B Fig, respectively).