Quantifying Phagocytic Uptake of Diverse Substrates

Human α-synuclein preformed fibrils [26 ], myelin debris [27 ] and immunoglobulin G-opsonized red blood cells [28 (link)] were labelled with pHrodo™ Green STP ester or pHrodo™ Red succinimidyl ester (Thermo Fisher Scientific) as previously described and used at the following respective concentrations which were determined to be non-saturating: 1 μM, 15 μg/mL and 5 × 10⁴ cells/mL. Bioparticles of pHrodo™ Green-labelled Escherichia coli (E. coli) were purchased from Thermo Fisher Scientific and used at a concentration of 25 μg/mL. Cells were incubated with the labelled substrates for three hours unless otherwise indicated, and before being counterstained with Hoechst 33342 (5 μg/mL). Total green fluorescence intensity per cell was quantified on a CellInsight CX5 High Content Screening Platform. All conditions were assessed in triplicate. Unchallenged cells were used to measure background/autofluorescence. Histograms were generated using the Python library Matplotlib. Internalization of fluorescein isothiocyanate (FITC) -labelled myelin was assessed similarly, except cells were washed with 0.4% trypan blue solution to quench extracellular fluorescence prior to imaging.