Anti human tnfα apc

In order to detect TNFα cytokine secretion, cells were coated with catch antibodies (Miltenyi Biotec) before the polarization period. After polarization, both the in-bulk and in-droplet cultured cells were obtained for staining and flow cytometric analysis. Cells were stained for viability using Zombie NIR™ (Biolegend) following the manufacturer’s protocol with a total dilution of 1:5.000 in PBS. Subsequently, cells were stained using a cocktail of fluorescent antibodies for surface markers and bound cytokines; anti-Human cluster of differentiation 80 (CD80)- APC-R700 (BD Bioscience), anti-Human C-C chemokine receptor 7 (CCR7) - Brilliant violet 421™, anti-Human CD206- PE/Cy7, anti-Human CD200R- PE/Dazzle™ 594, (all from Biolegend) and anti-human TNFα-APC (Miltenyi Biotec). Antibodies were titrated and used at a dilution of 1:40 for optimal performance. Flow cytometric measurement was performed using FACSaria III (BD bioscience) and a total of 10.000 total events were measured per sample. Results were analyzed using FlowJo (FLowJo LLC), which was also used for generating histograms and dot plots. Marker expression was quantified using Median Fluorescent Intensity (MFI). Results were plotted using Prism8 (GraphPad software).

For detection of cytokine secretion cells were coated with capture antibodies for IL-10 and TNFα (Miltenyi Biotec) prior to macrophage stimulation. After stimulation cells from bulk or droplet cultures were collected and stained with Zombie NIR™ (Biolegend) at a final dilution of 1:2.000 to check viability. After washing cells were stained using an antibody cocktail of the following fluorescent antibodies: anti-Human cluster of differentiation 80 (CD80)- APC-R700 (BD Bioscience), anti-Human C-C chemokine receptor 7 (CCR7) - Brilliant violet 421™, anti-Human CD206- PE/Cy7, anti-Human CD200R- PE/Dazzle™ 594, (all from Biolegend), anti-human TNFα-APC and anti-human IL-10-PE (both from Miltenyi Biotec). Flow cytometric analysis was performed using FACScanto II or FACSaria III (both from BD bioscience). Results were analyzed using FlowJo (FlowJo LLC). Marker expression was quantified using median fluorescent intensity (MFI).