Journal: bioRxiv

Article Title: Mucin MUC5B levels are reduced in the colonic epithelia of ulcerative colitis patients

doi: 10.1101/2024.02.21.581373

Figure Lengend Snippet: (A) Scoring system to analyze MUC5B proteins in intestinal tissues. Positively stained cells were quantified per field of view under 10X magnification. At least five fields of view were assessed for each tissue and staining patterns were scored as follows: 0 (negative or very low numbers of positive cells, 0-10%), 1 (low, 10-25%), 2 (moderate, 25-50%) or 3 (high, >50%). (B) Immunohistochemical analysis of MUC5B in an inflamed colonic segment from a UC patient. (C) Quantification of MUC5B staining in colons from non-IBD controls (n=12) and in inflamed colons from UC patients (n=28). Error bars represent SEM, * P < 0.05.

Article Snippet: The plates were incubated at 37°C for 20 min to allow the domes to solidify after which 750 μl of human intestinal organoid growth media (Human IntestiCult Organoid Growth Medium, hIOGM, StemCell Technologies, 06010) was added along with 10 μM ROCK inhibitor (Y-27632, Miltenyi, 130-106-138).

Techniques: Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Representative 3D confocal images of WGA-Alexa Fluor 488 and phalloidin–Texas Red (F-actin labeling) co-stained organoids with apical-out (AO), apical-basal (AB) and basal-out (BO) topology. Scale bar is 100 µm. B: Large field mosaic scan of PFA-fixed pig intestinal organoids after 18 h of polarity reversion, co-stained with fluorescent WGA and phalloidin. Left: representative images of AO, AB and BO organoids, indicated on mosaic image ( right ). Scale bar is 100 µm. C: Comparison of the yield of AO, AB and BO topology, estimated with WGA staining and F-actin labeling, respectively (AO organoids: 93.1% / 95.5%, BO organoids 0.53% / 0.55%, analyzed from 4 mosaic scanned images, see table ST3). D: Total quantification of topology, observed with WGA and Nile Red labeling for polarity reverted organoids: AO with lipid droplets (AO LDs) and AO without lipid droplets (AO no-LDs), AB and BO, quantified from mosaic scanned image. E: Lipid droplets display a characteristic distribution in AO organoids, contrasting with BO. F: Co-staining with WGA and Nile Red reveals organoid structure in apical-basal organoid (AB). Scale bar is 50 µm.

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Labeling, Staining, Comparison

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Scheme of experimental workflow. B: Representative confocal fluorescence images and corresponding phasor plots of the pig small intestinal organoid incubated with NP A-D (10 µg/ mL, 18 h), co-stained with Nile Red. NP A, B and D displayed signals on both fluorescent intensity images and phasor plots, whereas type C showed no signal, similar to control. C: Different types of uptake of NP type D uptake (10 µg/mL NPD, 20% laser power) into organoids, co-stained with WGA, with respect to the topology and size. Left: partial NP D uptake in AO, showing high pixel signal on a phasor plot. Middle: organoid size-dependent NP D uptake in AO. ROI 1: enhanced NP D pixel signal in a small AO, ROI 2: reduced NP D pixel signal in larger organoid. Right: Homogeneous NP D distribution in BO with strong signal on a phasor plot. D: Topology-dependent uptake of NP D (magenta) in apical-basal organoid (AB), co-stained with WGA (green) and Nile Red (yellow). Left: 3D reconstruction shows distinct NP D distribution in AO and BO regions. Right: representative fluorescence image of AB organoid taken from 3D reconstruction. AO and BO regions defined by WGA and Nile Red signals, with corresponding NP type D phasor FLIM plots of these regions (bottom right).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Fluorescence, Incubation, Staining, Control

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A, B: Representative images of NP D uptake (10 µg/mL, 18 h) in pig intestinal organoids in respect to their apical-basal topology with high loading in both AO and BO (A) and high loading in BO and zero at AO (B). Scale bar is 50 µm. C, D: Comparison of NP D uptake in AO vs. BO organoids as a function of loading concentration (0-1 µg/mL, left panel and 1-50 µg/mL, right panel) on a widefield fluorescence microscope (intensity-based approach, C), and confocal FLIM microscope (phasor FLIM event counting approach, D). FLIM events were counted from the phasor plots reconstructed in the napari phasor plugin from the exported list of G and S coordinates. Results of one of the two independent experimental replicates are shown. Both C and D data were produced from the same samples. The box charts represent 25, median and 75 percentiles with dots corresponding to individual intensity or event count square ROI square ROI normalized values. Attribution of organoids to AO and BO-topology groups was done based on WGA staining. BO group also includes AB organoids. Statistical comparison between AO and BO groups over a range of loading concentrations was performed by Mann-Whitney test (lines represent detected statistical difference, p <0.05).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Comparison, Concentration Assay, Fluorescence, Microscopy, Produced, Staining, MANN-WHITNEY