Extended Data Fig. 10: Flow cytometry gating strategy to determine total cell counts.
From: The Parkinson’s disease drug entacapone disrupts gut microbiome homeostasis via iron sequestration
A fixed staining–gating strategy was used. a. Gating was performed on the Syto9-Alexa488-A channel to discriminate between debris or background and microbial events stained with SYTO™9. Gating was established by comparing colour density acquisition plots of the sample unstained (top panel) with plots from the same sample stained with SYTO™9 (bottom panel). Each panel reflects over 100,000 registered events. b. An additional gating was performed on the CountBright-Cy5-A channel to count in parallel the number of CountBrightTM beads (added to the sample). Gating was established by comparing acquisition plots of the sample without the addition of beads (top panel) with plots from the same sample with CountBrightTM beads added (bottom panel). Events pseudocolored red represent CountBrightTM bead events that fall within the FSC-H/CountBright-Cy5-A gate. The number of cell events were defined as events registered in the FSC-A/Syto9-Alexa488-A gating area, excluding number of bead events observed in the FSC-H/CountBright-Cy5-A gate (as CountBrightTM beads also display Alexa488-A signal). Concentration of cells in a sample was calculated as: number of cell events divided by the number of bead events, and then multiplied by the known concentration of beads in the sample, according to the assigned bead count of the lot. At least 2,000 bead events were recorded per sample, for accurate counting. The acquisition rate did not exceed 2,000 events per second.
