human cd3 positive selection kit  (Miltenyi Biotec)

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: Assessment of the functional impact of neutrophils from Ax-3 crew on T cell responses (A) CD3 + T cells isolated from the peripheral blood of the astronauts before and after the mission were stimulated with anti-CD3/28 beads and proliferation responses were measured by a CFSE-based flow cytometric assay. Histograms obtained from an astronaut are shown as a representative result. T cells from healthy volunteers (ground controls) were also studied (gray histogram). (B) A schematic (created in BioRender Created in BioRender. Esendagli, G. (2025) https://BioRender.com/a4m1kdr ) is presented for the experimental setup used for testing the functional impact of neutrophils. NDN and LDN cells collected from astronauts before (L-1d) and after (R+1day) the Ax-3 mission were co-cultured with the anti-CD3/CD28-stimulated T cells obtained from healthy control individuals. T cells proliferation (C), and secretion of IL-2 (D) and IFN-γ (E) were determined following incubation for 72 h. Statistical analyses were performed with paired t test (∗∗ p ≤ 0.01). 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: CD3 + T cells were isolated from peripheral blood samples of healthy donors by using Human CD3 Positive Selection Kit (Miltenyi).

Techniques: Functional Assay, Isolation, Flow Cytometry, Cell Culture, Control, Incubation

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: Comparison of the impact of space flight and suborbital flight on immunophenotypic and functional characters of NDN (A) Granularity (SSC) and size (FSC) values of NDN before and after the Ax-3 mission and the suborbital flight mission were quantified by flow cytometry. (B–D) Expression levels of the neutrophil surface molecules were calculated by normalizing the median fluorescence intensity (MFI) to AF for each marker and a comparative heatmap was prepared (A.U., arbitrary units). The change in ROS (C) and NO (D) production capacities in NDN after the Ax-3 mission and the suborbital flight was calculated by comparing the data of the NDN obtained before the mission. (E–H) NDN cells collected from the astronauts before and after the Ax-3 and suborbital flight missions were co-cultured with the anti-CD3/CD28-stimulated T cells from healthy control individuals. T cell proliferation in the co-cultures with increasing amounts of NDN was normalized to that of the T cells stimulated alone. In addition, the changes in T cell proliferation (F), and secretion of IL-2 (G) and IFN-γ (H) in the presence of NDN after the Ax-3 mission and the suborbital flight were calculated by comparing to the data obtained with the NDN collected before the missions. Statistical analyses were performed with paired t test (∗∗ 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: CD3 + T cells were isolated from peripheral blood samples of healthy donors by using Human CD3 Positive Selection Kit (Miltenyi).

Techniques: Comparison, Functional Assay, Flow Cytometry, Expressing, Fluorescence, Marker, Cell Culture, Control

Journal: Science Advances

Article Title: Armored human CAR T reg cells with PD1 promoter-driven IL-10 have enhanced suppressive function

doi: 10.1126/sciadv.adx7845

Figure Lengend Snippet: ( A ) Schematic diagram of alloantigen-based suppression assay shown in (B) and (C). HLA-A2 + DCs were cocultured with HLA-A3 − T reg cells for 72 hours. Allogeneic CPD eFluor 450–labeled HLA-A3 + CD3 + responder T cells were then added, and cocultures were maintained for an additional 96 hours. ( B ) Representative CD4 + responder T cell proliferation, gated on live HLA-A3 + CPD eFluor 670 − CPD eFluor 450 + CD4 + cells. ( C ) % Suppression of CD4 + responder T cell proliferation, relative to responder T cells cultured without T reg cells. ( D ) Schematic diagram of islet antigen–based suppression assay shown in (F) to (H). HLA-A2 + HLA-DR4 + DCs were cocultured with T reg cells for 48 hours. CD4 + 4.13-TCR + responder T cells and 1 nM GAD65 peptide were then added, and cocultures were maintained for an additional 48 hours. ( E ) CD4 + 4.13-TCR + T cell proliferation following 96-hour coculture with immature HLA-DR4 + DCs and varying GAD65 peptide concentrations (without T reg cells). Control cells were pulsed with an irrelevant hemagglutinin peptide (100 nM). Division indices provided. ( F ) Representative CD4 + responder T cell proliferation, gated on live CD4 + CPD eFluor 670 − mTCRβ + cells. ( G ) % Suppression of responder T cell proliferation, relative to responder T cells stimulated without T reg cells. ( H ) Relative cytokine analysis from 1:128 T reg cell:responder T cell (T resp cell) ratio. TNFα, IL-17A & IL-17F were not detected. Averaged data are means ± SEM with non-linear regression lines ( n = 8 to 10). Statistical significance was determined using mixed-effects analysis with P values shown. n.s., not significant.

Article Snippet: After 3 days, CD3 + responder T cells (previously enriched from an HLA-A3 + individual using the EasySep Human CD3 Positive Selection Kit II; STEMCELL Technologies) were labeled with CPD eFluor 450 and added to the cocultures at a 5:1 responder T cell:DC ratio.

Techniques: Suppression Assay, Labeling, Cell Culture, Control

Journal: Experimental Hematology & Oncology

Article Title: Engineered circular RNA-based DLL3-targeted CAR-T therapy for small cell lung cancer

doi: 10.1186/s40164-025-00625-8

Figure Lengend Snippet: Optimization of electroporation conditions for circRNA expression in Human CD3 + T cells. A Comparison of EGFP expression in T cells electroporated with circRNA at different voltages (1400V, 1600V, and 1800V). B Flow cytometry analysis of anti-DLL3 CAR expression at varying electroporation voltages. C Evaluation of CAR expression and apoptosis rates in T cells electroporated with various concentrations of circRNA (1–6 µg/million cells). D Assessment of immunogenicity in circRNA and mRNA-transfected T cells under optimized electroporation conditions. E Assessment of CAR expression duration in circRNA and mRNA-transfected T cells. F Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CCK-8 assay. G Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CFSE assay. circRNA circular RNA, mRNA messenger RNA, EGFP enhanced green fluorescent protein, CAR-T chimeric antigen receptor T cell, DLL3 Delta-like Ligand 3, CCK-8 Cell Counting Kit-8, CFSE Carboxyfluorescein Succinimidyl Ester

Article Snippet: CD3 + T cells were then purified employing the EasySep TM Human CD3 Positive Selection Kit II (Stemcell, 17851).

Techniques: Electroporation, Expressing, Comparison, Flow Cytometry, Immunopeptidomics, Transfection, CCK-8 Assay, CFSE Assay, Cell Counting