Journal: International Journal of Molecular Sciences
Article Title: Not-So-Rare Defects of RBC Lipidic Composition: Four New Cases of Flippase Deficiency Due to ATP11C Mutations
doi: 10.3390/ijms26167722
Figure Lengend Snippet: Flippase activity of RBCs of patients with ATP11C mutations and their relatives. ( A ) Proportion of fluorescent phosphatidylserine (NBD-PS) transported into the inner leaflet in patients and family members after 20 min incubation (MFI NBD-PS + BSA/MFI loaded NBD-PS, colored lines). Dotted lines represent blocked NBD-PS internalization after NEM treatment. Values are mean n = 2 experiments for each sample. ( B ) Primary NBD-derived fluorescence data from flow cytometry. NBD-PS loaded onto erythrocyte membranes for 20 min. Stars indicate the population of the NBD-PS transported defective cells. MFI: Mean fluorescence intensity; BSA: bovine serum albumin. NEM: mM N-ethylmaleimide; F1-P1: Family 1, Patient 1; F1-P2: Family 1, Patient 2; F1-Mo: Family 1, mother; F1-Fa: Family 1, father; F2-P1: Family 2, Patient 1; F2-Mo: Family 2, mother; F2-Fa: Family 2, father; F3-P1: Family 3, Patient 1; F3-Mo: Family 3, mother; F3-Fa: Family 3, father.
Article Snippet: Briefly, 10 μL of 0.1 mg/mL Fluorescent PS (16:6-06:0 NBD-PS, Avanti Polar Lipids, Inc., Albaster, AL, USA) was added to 1 mL of washed erythrocytes resuspended at 5% hematocrit in PBS with 0.2% glucose (PBS-G); samples were incubated for 0–20 min at 37 °C, and every 5 min, 20 μL of the erythrocyte suspension was washed with 1 mL PBS-G with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MA, USA) to remove remaining NBD-PS, such that the remaining fluorescence represented PS that flipped to the inner leaflet.
Techniques: Activity Assay, Incubation, Derivative Assay, Fluorescence, Flow Cytometry
