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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 - <t>CD66b</t> + 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).
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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 - <t>CD66b</t> + 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).
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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 - <t>CD66b</t> + 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).
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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 - <t>CD66b</t> + 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).
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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).

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