Immune cell evaluation by flow cytometry
LYSED
From each blood sample, whole blood was used to evaluate changes to immune cell numbers and activation status. All immunostaining was performed in triplicate on fresh blood within an hour of drawing the blood. Three different panels of monoclonal antibodies were used: 1) NK panel: A panel focused on NK cells, NKT cells, T cells, and monocytes and activation markers on those cell types, 2) T cell panel: A panel focused on CD8+ versus CD4+ T lymphocytes and also evaluating CD45 isoforms on each subset of T cells as well as B lymphocytes, and 3) A gamma delta (γδ) T cell panel, focused on γδT lymphocytes and activation markers on those cells.The NK cell panel made use of the following monoclonal antibodies: CD3-SuperBright702 (clone OKT3), CD25-SuperBright600 (clone BC96), CD56-Phycoerythrin (clone CMSSB), CD57-eFluor450 (clone TB01), and CD69-Fluorescein isothiocyanate (clone FN50).The T cell panel made use of these monoclonal antibodies: CD4-SuperBright702 (clone SK3), CD8-Fluorescein Isothiocyanate (clone OKT8), CD19-SuperBright600 (clone SJ25C1), DC45RA-SuperBright436 (clone HI100), and CD45R0-Phycoerythrin (clone UCHL1).The γδT cell panel made use of these monoclonal antibodies: γδT cell antibody-Fluorescein isothiocyanate (clone SK7), CD5-SuperBright702 (clone UCHT2), CD25-Brilliant Violet421 (clone 2A3), CD56-SuperBright600 (clone TULY56), and CD69-Phycoerythrin (clone FN50).For the NK and γδT cell panels, triplicate samples of 100 μL heparinized whole blood were stained with the mix of monoclonal antibodies listed above, incubated for at least 15 minutes at room temperature in the dark, followed by addition of 2 mL of High-Yield Lysing buffer, mixing, and incubation for at least 10 minutes at room temperature in the dark. Samples were transferred to 2-mL deep-well 96-well plates and samples were acquired by flow cytometry within 4 hours of staining.For the T cell panel, triplicate samples of 100 μL heparinized whole blood were stained with the mix of monoclonal antibodies listed above, incubated for at least 15 minutes at room temperature in the dark, after which 100 μL Cal-Lyse buffer was added and the samples were mixed carefully. After another 10 minutes of incubation the red blood cells were lysed by addition of 1 mL of distilled water and mixed by vortexing. Samples were stored in the dark and acquired by flow cytometry within 24 hours.All samples were acquired using an acoustic-focusing Attune™ Nxt flow cytometer (Thermo Fisher Scientific), using a microplate-based Autosampler. Data analysis utilized gating on forward/side scatter to identify lymphocyte and monocyte populations, followed by gating based on NK and T cell markers. Each cell subset was then analyzed for expression levels of the activation markers.