Fig 1.
Effects of monolayer plating density on cell proliferation.
Reconstructed whole well images of monolayers from a jejunal HIO line plated with varying cell densities and cultured for two days in differentiation medium or proliferation medium (L-WRN conditioned medium). HIOs were then pulsed with EdU for 24 hours and EdU-stained to label proliferating cells (green), then stained with DAPI (blue) to label remaining nuclei. Images were taken at 4 × magnification (scale bar = 1000 μm).
Fig 2.
CV8000 image reconstruction and postprocessing. A) The whole well of an HIO monolayer was imaged on a CV8000 confocal by obtaining nine images of the well at 4 × magnification. Images were postprocessed and partial-well images were stitched together in CellPathfinder to reconstruct the whole-well image. B) To avoid edge effects, a central crop area was obtained from each reconstructed whole-well image by using the CellProfiler crop function before quantification.
Fig 3.
Pipeline identification of varying donor epithelial responses to microbial supernatants.
HIO monolayers from three infant jejunal lines were treated with cell-free supernatants from Ligilactobacillus salivarius 112 (LS112) or Lacticaseibacillus rhamnosus 105 (LR105) grown in differentiation medium (Diff). Control monolayers were treated with Diff. Monolayers were pulsed with EdU for 24 hours. A) Monolayers were stained for EdU (green) to label proliferating cells and DAPI (blue) for cell nuclei. Images were captured using a CV8000 confocal at 4 × magnification (scale bar = 500 μm). B) EdU and DAPI-stained areas of each well were quantified via pipeline analysis of pixel staining and normalized for cell density. The percentage of green area was compared between supernatant-treated and Diff-treated wells using a one-way ANOVA. C) EdU-positive cells were quantified by flow cytometry. The percentage of EdU-positive cells was compared between supernatant-treated and Diff-treated wells using a one-way ANOVA. D) Correlation plots of pipeline-quantified percentage green area values averaged across the technical replicates plotted against the averaged flow cytometry EdU-positive values.
Fig 4.
Chromagranin A staining in a neurogenin-3 inducible HIO line.
Three monolayers of an inducible neurogenin-3 jejunal HIO line were cultured with differentiation medium. Three wells were induced with doxycycline to increase enteroendocrine cells (EECs) and three were not induced. A) Chromagranin A-stained and DAPI-stained areas of each well were quantified via pipeline analysis of pixel staining. The percentage green area was compared between low EEC (non-induced) and high EEC (induced) wells using an unpaired Student’s t-test. B) Monolayers were stained for chromagranin A (green) for EECs and DAPI (blue) for cell nuclei. Monolayers were imaged on a CV8000 confocal at 4 × magnification (scale bar = 500 μm). c, Chromagranin A-positive cells were quantified by flow cytometry. The percentage of Chromagranin A-positive cells was compared between low EEC (non-induced) and high EEC (induced) wells using an unpaired Student’s t-test.