Journal: iScience

Article Title: Spaceflight alters the immune regulatory functions of neutrophil granulocytes on T lymphocytes

doi: 10.1016/j.isci.2025.114380

Figure Lengend Snippet: Immunophenotypic analysis of peripheral blood neutrophil granulocytes from axiom mission 3 (Ax-3) crew (A) A schematic illustrating the time points of blood draw and layout of the 18 days long (18days) Ax-3 mission to International Space Station (ISS). Preflight blood samples were collected on one day before the launch (L-1d) and postflight blood samples were collected on one day after the return (R+1day). (B) Peripheral blood was subjected to density gradient centrifugation and normal-density neutrophils (NDN, >1.077 g/mL)) and low-density neutrophils (LDN, <1.077 g/mL) were isolated. This figure was created in BioRender (Esendagli, G. (2025) https://BioRender.com/a4m1kdr ). (C) Representative flow cytometry dot-plots of the <1.077 g/mL blood fraction are shown from an astronaut. Red population depicts the LDN cells determined after back gating the CD11b + HLA-DR - CD66b + cells found among the peripheral blood mononuclear cells (PBMC). (D) Percentage of LDN determined at preflight and postflight analyses. Each dot represents a member of the Ax-3 crew. (E) Granularity (SSC) and size (FSC) values of NDN and LDN were quantified by flow cytometry. (F) Flow cytometry offset histograms of neutrophil cell surface markers associated with cellular activation are demonstrated for a representative astronaut. Autofluorescence (AF) values were taken as technical control. (G) Percentage of NDN and LDN populations positive for CD11b, CD33, CD62L, CD80, CD86, and CD69 markers. (H) Expression levels of the neutrophil surface molecules was calculated by normalizing the median fluorescence intensity (MFI) to AF for each marker and a comparative heatmap was prepared (A.U., arbitrary units). Statistical analyses were performed with paired t test (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001). The p values calculated to be > 0.05 were not indicated. The data are presented as mean with standard error of mean (SEM).

Article Snippet: StraightFrom® Whole Blood CD66b MicroBeads, human , Miltenyi , Cat # 130-104-913.

Techniques: Gradient Centrifugation, Isolation, Flow Cytometry, Activation Assay, Control, Expressing, Fluorescence, Marker

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD16 + cultured neutrophils were similar to PMNs in mobility, ROS production, phagocytosis, and microbial killing (A) Boxplots and violin plots showing the relative expression of genes and proteins involved in neutrophil effector functions . (B) CD11b/CD18-mediated adhesion assay of PMNs (blue) and SL-II CD16 + neutrophils (pink) to uncoated plastic plates ( n = 4). (C) Chemotactic potential of fluorescently labeled PMNs (blue) and SL-II CD16 + neutrophils (pink) based on movement through filters with a pore size of 3 micron ( n = 5). (D) NADPH oxidase assay to determine the production of extracellular peroxide after the addition of opsonized E. coli , zymosan, STZ, PMA, and PAF/fMLP of PMNs (blue) and SL-II CD16 + neutrophils (pink) ( n = 4 for SL-II CD16 + neutrophils and n = 8 for PMNs). (E) Phagocytosis of either unopsonized or opsonized zymosan by PMNs (blue) versus SL-II CD16 + neutrophils (pink) measured by flow cytometry ( n = 3 for SL-II CD16 + neutrophils and n = 4 for PMNs). (F) Representative images of phagocytosis of either unopsonized or opsonized zymosan labeled with FITC solution (green) by PMNs labeled with calcein red-orange (orange) versus SL-II CD16 + neutrophils labeled with calcein red-orange (orange) at the latest timepoint as visualized by imaging flow cytometric analysis. Scale bar was set at 10 μm ( n = 3). (G and H) Killing of opsonized E. coli and S. aureus, and unopsonized and opsonized C. albicans, respectively, shown for PMNs (blue) ( n = 3) versus SL-II CD16 + neutrophils (pink) ( n = 3). Killing was quantified as the inverse of colony-forming units (CFU) as a percentage relative to the CFU at the start of the assay. Negative values, signifying an increase in the number of colonies were considered to be 0. Data shown in (A–D, H) is represented as median and interquartile range, and (E) and (G) is represented as mean ± SD. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05 and ∗∗ p < 0.01. n values represent the number of individual donor samples.

Article Snippet: Mouse Anti-Human CD66b Monoclonal antibody, FITC Conjugated (Clone 80H3) , Bio-Rad , Cat #MCA216F; RRID: AB_2077860.

Techniques: Cell Culture, Expressing, Cell Adhesion Assay, Labeling, Pore Size, Flow Cytometry, Imaging, MANN-WHITNEY