Cd56 bv421

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.

Dendritic cell-enriched cell suspensions were stained with an antibody cocktail consisting of the following antibodies in PBS + 2% human sera for 30 min on ice: CD1c-APC/Cy7 (L161; BioLegend), CD3-BUV395 (UCHT1; BD Bioscience), CD11b-A700 (M1/70; BioLegend), CD11c-PE/Cy7 (3.9; BioLegend), CD14-A700 (HCD14; BioLegend), CD19-V450 (HIB19; BD Bioscience), CD20-eFluor 450 (2H7; eBioscience), CD56-BV421 (5.1H11; BioLegend), CD123–BV605 (6H6; BioLegend), CD141-BV711 (1A4; BD Bioscience), CD303-PerCP/Cy5.5 (201A; BioLegend), HLA-DR-PE-CF594 (G46-6; BD Bioscience), and NKp46-BV421 (9E2; BioLegend). After washing the cells, they were resuspended in PBS + 2% human sera + 0.1 mg/ml 4′,6-diamidino-2-phenylindole and cell sorted using a BD FACSAria II cell sorter into CD1c⁺ DCs (CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA–DR⁺CD11c⁺CD1c⁺), CD141⁺ DCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11c⁺CD141⁺), pDCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻NKp46⁻HLA-DR⁺CD123⁺CD303⁺), and monocytes (CD3⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11b⁺CD14⁺). The purity of sorted cell populations was reanalyzed and routinely above 95%.

BAL cells (200,000–500,000 cells) and PBMC (100,000–200,000) were resuspended in Facs Buffer (10%FBS+4mM EDTA +PBS) and stained for 20 minutes at room temperature with the following antibodies used per manufacturer’s instructions: CD3-FITC (Biolegend, #344804), CD4-APC-Cy7 (BD Biosciences, #BD557871), CD8-APC (Biolegend, #344722), CD56-BV421 (Biolegend, #362552), Gamma-delta-PE (Biolegend, #331210), CCR7-PECY7 (Biolegend, #353226) and CD45RA-PECY5 (Biolegend, #304110). Compensation was performed using antibody capture beads (anti-mouse k; BD Biosciences, #552843). Zombie Aqua fixable viability kit (BioLegend, #433101) was used as the live/dead discriminator. Cells were fixed, acquired and analyzed on a MacsQuant flow cytometer using MacsQuant software version 2.11.176.19438. Analysis was performed using Flow Jo version 10.6 (Oregon, USA). Cells where gated on lymphocyte sized, singlet, live cells. BAL and PBMC flow cytometric counts and percentages for each participant can be found in the S2 Dataset.