FIGURE 4.

Miniaturisation of air–liquid interface (ALI) cultures to 96-well format to generate primary ciliary dyskinesia (PCD) patient models. a) Schematic showing experimental set-up for immunofluorescence screening in 96-well transwell ALI system. The image in the two-dimensional culture is taken from figure 1b. b) Immunofluorescence images demonstrating the presence of β-tubulin-expressing cilia (green) and MUC5AC-expressing mucosecretory cells (red) in 96-well format ALI cultures. Two PCD donors, one with static cilia (PCD 1) and one with circular cilia (PCD 5) defects, are shown in comparison to a healthy donor control. Images are representative of two healthy donors and four PCD donors tested. Scale bar: 50 µm. c) Ciliary beat frequency analysis comparing multiple recordings from two healthy donors (Healthy 3 and 4) and two PCD patients (PCD 5 and 12) in 24-well and 96-well formats. Data points are individual recordings from four to five wells, with five videos recorded per well. p-values were derived using a two-way ANOVA with Holm–Sidak's test for multiple comparisons, with statistical significance set at p<0.05. ns: nonsignificant. **: p<0.01. d) Ciliary beat pattern analysis in 96-well format. 24-well equivalent values are found in table 1.