cd3 microbead positive selection  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Early-life mucosal T cells direct intestinal stem cell fate via a coordinated developmental program

doi: 10.64898/2026.05.08.723752

Figure Lengend Snippet: (A) Co-culture strategy (created with Biorender.com ). Small intestine (SI) was cryopreserved, and stable organoid lines were generated. T cells were isolated from SI tissue (B-E, H-K) or cord blood (F-G) by positive selection. CD3+ cells were co-cultured with established SI organoid lines generated from fetal (B-I) , neonatal (D-E) , or adult (I-K) donors. (B, D, F, H) Representative images from day 6 of co-culture. Scale bar is 530 µm. (C) Data were generated from one organoid and T cell donor per fetal age group and analyzed by one-way ANOVA with Tukey’s post-hoc test. Only significant comparisons are denoted. Data are shown as means ± SEM. (E, G, I) Data were generated from 2-4 organoid and T cell donors each. Data were analyzed via Mann-Whitney and are shown as medians ± IQR. (K) Data were generated from 3 organoid donors and 4 T cell donors and are normalized to the number of organoids generated in mock (no T cell) conditions, represented as fold change. Each dot represents the average of three technical replicate wells (C) or one well of organoids (E, G, I, K) . Data were analyzed via Welch’s t-test and are shown as means ± SEM. Each experiment was repeated 2-3 independent times. Each dot represents one well of organoids. Significance is indicated as follows: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns, not significant (p>0.05).

Article Snippet: T cells were isolated via CD3 microbead positive selection (Miltenyi, cat#130-050-101 or 130-097-043) via MS or LS columns (Miltenyi, cat#130-042-201 or 130-042-401) per manufacturer’s instructions (Miltenyi).

Techniques: Co-Culture Assay, Generated, Isolation, Selection, Cell Culture, MANN-WHITNEY

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: bioRxiv

Article Title: Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

doi: 10.1101/2025.10.09.681473

Figure Lengend Snippet: (A) Allele plot showing off-target sites identified using GUIDE-Seq. K562 cells were electroporated with SpCas9-sgRNA vector targeting intron 2 of ATG5 and GUIDE-Seq dsDNA, and genomic DNA was harvested three days post-electroporation. Dots represent matches with the target sequence, while mismatches are colored. (B) Same as (A) but for CD3+ T cells electroporated with SpCas9 RNPs targeting ATG5 and GUIDE-Seq dsDNA. Genomic DNA was harvested on Day 3 (top) or Day 11 (bottom) post-electroporation. Day 3 read counts are from one of two different healthy donors. Day 11 read counts are from one healthy donor. (C) Indel quantification as determined by CRISPResso2 analysis from targeted amplicon sequencing of genomic DNA from T cells electroporated with SpCas9 or HiFiCas9 RNPs. The dotted line indicates a 0.1% limit of detection. Results are from n = 2 independent experiments performed with T cells from two different healthy donors.

Article Snippet: CD3+ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs; STEMCELL) using positive selection CD3 microbeads (Miltenyi).

Techniques: Plasmid Preparation, Electroporation, Sequencing, Amplification