Additional file 1 of Neutrophil degranulation and severely impaired extracellular trap formation at the basis of susceptibility to infections of hemodialysis patients
Creators: name: Talal, Salti
name: Mona, Khoury
name: Karem, Awad
name: Yaniv, Lerenthal
name: Reut, Harari-Misgav
name: Ariel, Shemesh
name: Moran, Avraham-Kelbert
name: Harel, Eitam
name: Campisi-Pinto, Salvatore
name: Mahmoud, Abu-Amna
name: Raul, Colodner
name: David, Tovbin
name: Gil, Bar-Sela
name: Idan, Cohen
Publisher: name: figshare
Year: 2022
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Marine Biology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Fig S1. Comparison of neutrophil isolation methods shows the superiority and advantage of negative selection cell sorting. (A) Flow cytometry analysis of cell pellets population separated using dextran, Max exp, or Stem Cell kits. The left column show neutrophil content of lysed whole blood start sample. The Center column shows the final isolated cell pellets of CD16+ cells, and the right column shows an analysis of isolated cell pellets with CD16 and CD66b. The CD16high CD66bhigh cell population is defined as neutrophils, and CD16low CD66bhigh as eosinophils. (B) Yields and kits efficiency of isolated neutrophil cells per ml blood. (C) Representative measurements of neutrophils' baseline activation result from the isolation/sorting technique. The extent of extracellular ROS release assessed baseline activation before and after stimulation with PMA. Fig. S2. (A) Distributions of gene type annotations of differentially expressed genes in HD Upregulated genes (DEG_UP) and HD downregulated genes (DEG_Down). (B) Heatmap of significant differentially expressed HD upregulated genes on all 14 HC and HD samples. The expression level is normalized from 0 (no expression) to 1 (highest expression). Fig. S3. No difference in uninduced neutrophil cells of HC and HD patients over 4h. Image analysis of uninduced (with no stimulation) neutrophil cells n=6 donors from each group (e.g., HC or HD). HC and HD cells showed no difference in the distribution of nuclei architecture or any signs of spontaneous NETosis after Incubation of 4h. Neutrophils taken from HD patients and stimulated with LPS generated less intracellular free radicals than HC donors. (B) live-cell measurement of global ROS levels and superoxide in neutrophils induced with 100ng/ml LPS. Profiling ROS formation by fluorescence microscopy was performed with highly pure neutrophils from HC and HD patients, loaded with ROS/Superoxide detection reagents, and activated with LPS. General oxidative stress levels were monitored in the green channel, while the orange/Red channel detected superoxide production. Snapshots were taken in indicated time points (e.g., 0, 15, 45 min). (C) Neutrophils were isolated from whole blood samples of HC and HD. The kinetics of extracellular ROS release was performed with L-012 (20mM) with or without PMA (100nM) stimulation. The kinetics of extracellular ROS release was measured for 30 min using an ELISA plate reader. Results are presented as means ± S.E. of RLU of six independent donors from each group. Neutrophils induced with PMA were a positive control for extracellular ROS release.