FIGURE 1.

Neutrophils from subjects with R117H mutation show no apoptosis defect but ivacaftor treatment induces changes in activation and adhesion markers after 7 days of therapy. a) Total peripheral granulocytes isolated by Ficoll Paque gradient in SepMate tubes and hypotonic RBC lysis before treatment and following 2 and 7 days of treatment. b) Percentage of granulocyte cell types isolated demonstrate high purity neutrophils. c) Percentage of viable neutrophils, 6 and 24 h after isolation in R117H subjects (pre-treatment and following 2 and 7 days of treatment) compared to untreated cystic fibrosis (CF) with type I–III mutations and non-CF controls (R117H, n=7–10; CFI–III, n=4; non-CF, n=6). d) Gating strategy for neutrophil flow cytometry. Granulocytes were enriched from Ficoll pellet after RBC hypotonic lysis. Neutrophils were gated by FSC×SSC, singlets were identified by FSC-A×FSC-H and neutrophils by CD66b+ CD14- Ab staining. e) Extrinsic cell death receptor expression measured by flow cytometry and shown as mean fluorescence intensity (MFI). f) Morphological structure of isolated neutrophils (10× magnification). Black arrow signifies apoptotic cell. g) Neutrophil activation and adhesion marker expression. Individual markers analysed by t-test. h) Markers of neutrophil granules. Individual markers analysed by t-test. i) Correlation of change in neutrophil CD88 expression (MFI) with change in patient sweat chloride from day 0–7 analysed by linear regression. j) Correlation of change in neutrophil CD54 expression (MFI) with change in patient forced expiratory volume in 1 s (FEV₁) % predicted. All data are shown as mean±sem. Statistically significant values designated by asterisks. *: p<0.05 **: p<0.01 ***: p<0.001.