Anti cd32 pe

The analyses of the second cohort (Table 2) included evaluation of monocyte and granulocyte subpopulations. Immune cell subpopulations were analyzed by flow cytometry after FACS staining which was performed within 1 h of blood withdrawal on day of admission and day 1, 3 and 5 post stroke (LSRII, BD Bioscience) [anti-HLA-DR Alexa Flour 488; clone: L243; anti-CD11b Brilliant Violet 421, clone: LM2; anti-CD14 PerCP/Cy5.5, clone: 63D3; anti-CD16 Brilliant Violet 650, clone: 3G8; anti-CD62Ligand PE-Cy7, clone: DREG-56; and anti-CD32-PE, clone: FUN-2 (Biolegend)]. Dead cells were excluded from analysis by the Zombie NIR TM Fixable Viability Kit (Biolegend). The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc.). The percentage of cells expressing a specific activation marker was determined as well as the amount of the specific marker per cell as defined by the mean fluorescence intensity (MFI). Fluorescence minus one controls (FMO) were used to distinguish different monocyte and granulocyte populations.

EDTA blood was sampled and processed within 2 h to examine monocytes and granulocytes. After a red blood cell lysis using ACK lysing buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA), cells were stained by Zombie NIR^TM Fixable Viability Kit (BioLegend^TM) on ice for 15 min to distinguish dead and alive cells, followed by a second staining with the different cell surface antibodies for 10 min on ice. Subpopulations were analyzed by flow cytometry (LSRII, BD Bioscience) [anti-HLA-DR Alexa Flour 488, anti-CD11b Brilliant Violett 421, anti-CD14 PerCP/Cy5.5, anti-CD16 Brilliant Violett 650, anti-CD62Ligand PE-Cy7, anti-CD32-PE (Biolegend)]. Death cells were distinguished by Zombie NIR TM Fixable Viability Kit (Biolegend). 200.000 events were gathered per single cell gate.
The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc.). The percentage of cells expressing a specific activation marker was determined as well as the amount of the specific marker on cell surface as defined by the mean fluorescence intensity (MFI). For the differentiation of monocytes and granulocyte subpopulation as well as activation marker fluorescence minus one controls (FMO) were used. CD14^dim monocytes and CD16^dim neutrophil population was distinguished by gating the 25th percentile of main monocyte and neutrophil population, respectively (see Supplementary Figures 1, 2 for gating strategy).

ImageStream cytometry analysis of HIV-1_iGFP/JR-FL internalization was performed as previously described [19 (link)]. In brief, after conducting the ADP assay as above, THP-1 cells were resuspended at a final concentration of 5 x 10⁶ cells/mL. Using an ImageStreamX Mark II Imaging Flow Cytometer (EMD Millipore), fluorescent-virus images were collected in channel 2 (480-560nm) at 40x magnification. Internalization of positive events was measured by applying the ImageStream IDEAS Internalization and Spot Wizard algorithms, which defined the internal area as a mask of erosion of 4 pixels into the brightfield perimeter of the cell. Cells with internalized virus were defined by two criteria: an internalization score greater than 0.3 and a Spot Value greater than 3, in order to exclude surface-bound virus and background fluorescence, respectively.
ImageStream cytometry was also used to visualize internalization of HIV-1_iGFP/JR-FL used to decorate the surface of CEM.NKR-CCR5 cells. THP-1 cells were surface stained with anti-CD32-PE (BioLegend), and PE+/GFP+ images were collected in channels 2 and 3 (560–595 nm). Focused, single cells (based on gating on gradient root mean square, area, and aspect ratio of the brightfield image) were analyzed.