ALI paper figures ready.zip

(2020) ALI paper figures ready.zip. Zenodo doi:10.5281/zenodo.4107149 [Dataset]

Record type: Dataset

Abstract

Figure 1: Fast Fourier Transform analysis of ciliary movement using HSVA data. Figure 1 legend: Fast Fourier Transform analysis (left) ‘colour map’ corresponding to ciliary movement detected by HSVA (using a 20x objective lens) on the surface of an ALI-culture (right). The colour scale (left to right) depicts increasing CBF from 0 Hz (black) to 25 Hz (white). Black pixels also represent a CBF measurement outside of the detection threshold (below 2 Hz or above 50 Hz). Normal mean CBF at 37oC is 11-20 Hz. Scale bar represents 100 µM. Figure 2: Flow diagram of diagnostic sample processing for ALI-culture by repeat HSVA and MDT outcomes. Figure 2 legend: 70 patients’ ex vivo samples evaluated by HSVA (as part of the whole diagnostic process) and their ALI-culture outcomes were followed. HSVA on ALI-culture either confirmed the original HSVA finding, resolved an originally equivocal HSVA or remained equivocal despite culture. In bold, the ALI-culture HSVA outcomes are shown by MDT outcome (‘PCD’/ ‘PCD highly-likely’; ‘PCD highly-unlikely’ or ‘equivocal’ pending follow up, repeat tests or further tests (genetics or additional IF for example). Figure 3: TEM of cilia in transverse section and CBF (by HSVA) in nasal samples and after ALI-culture. Figure 3 legend: Representative TEM images a) and b) of in vitro ALI-cultured cilia in transverse section, showing a “9+2” microtubular arrangement. Cilia have normal ciliary ultrastructure in a) a ‘PCD highly-unlikely’ subject (with 3% microtubular defects, 18% inner and 4% outer dynein arm defects quantified from 102 cilia), and b) a ‘PCD positive’ subject (with 11% microtubular defects, 47% inner and 99% outer dynein arm absence quantified from 302 cilia). Scale bar represents 100 nm. Dot plots c) demonstrates the mean CBFs (Hz) of 57 ex vivo nasal brushing samples compared to their matched in vitro ALI-cultures (Wilcoxon paired test P=0.03). Data from ex vivo samples without a matched ALI sample were excluded (n=13), which was due to 7 ex vivo samples with variable CBP, 3 failed ALI-cultures and 3 not cultured. Figure 4: Characteristics of in vitro ALI-cultures derived from frozen liquid nitrogen storage. Figure 4 legend: a) There was no difference in the mean (±SD) TEER (Ohmes.cm2) of n=10 ‘PCD highly-unlikely’ and n=4 ‘PCD highly-likely’ ALI-cultures, recovered from liquid nitrogen cryostorage (post-LN2) (t test), or compared to n=4 healthy donor samples (non-frozen) controls (Mann Whitney test); measured in triplicate per transwell at 4 weeks ALI-culture, when cells were widely ciliated. b) The mean CBF (Hz) of n=4 matched PCD clinic samples differed before (ex vivo) and after liquid nitrogen storage (in vitro ALI-culture) P=0.01 (paired T test), but remained in UHS normal range 11-20 Hz. c) A representative SEM image from a ‘PCD highly-unlikely’ ALI-culture showing typical ciliation at week 4 post-LN2. d) Representative PCD diagnostic immunofluorescence [15] images from an SP8 laser scanning confocal microscope, showing a PCD clinic ALI-culture after cryostorage with 4% paraformaldehyde fixation and immunofluorescence labelling with anti-alpha-tubulin (cilia marker-Alexa488 secondary antibody, green), anti-RSPH4a (radial spoke head protein-Alexa549 secondary antibody, red) and DAPI (nuclei DNA stain, blue). Scale 20 µm.

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