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: 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

Journal: bioRxiv

Article Title: The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces

doi: 10.1101/2024.01.31.578101

Figure Lengend Snippet: a , CD3+ T-cells were isolated from 3 separate C57BL/6 mice and treated for 4 hr with UCM, SCM, or ACM to observe early changes in gene expression via RNA-sequencing. PCA plot shows reproducibility between replicates. b , Volcano plots showing genes in SCM- or ACM-treated cells with a >2-fold change in expression compared to UCM. c , Venn Diagram showing overlap between genes that are upregulated or downregulated in SCM- and ACM-treated cells. d , Hierarchical clustering of 17,486 genes with an expression signal greater than zero in any condition. Cluster 1 = genes upregulated in SCM, Cluster 2 = genes downregulated in ACM, Cluster 3 = genes with a similar loss of expression in SCM and ACM, Cluster 4 = genes with increased expression in ACM. e , T-cell activation-specific targeted gene analysis. n = 3 independent animals.

Article Snippet: Murine CD4+ and CD8+ positive selection kits (# 130-117-043, 130-117-044), human CD3+ positive selection kit (# 130-050-101), and murine CD3+ and CD8+ negative selection kits (# 130-095-130, # 130-104-075) were purchased from Miltenyi Biotec.

Techniques: Isolation, Gene Expression, RNA Sequencing, Expressing, Activation Assay

Journal: bioRxiv

Article Title: The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces

doi: 10.1101/2024.01.31.578101

Figure Lengend Snippet: a , Schematic of a DNA-based tension probe presenting α-CD3ε. b , Representative images and quantification of 4.7 pN tension signal exerted by polyclonal CD4+ and c , CD8+ T-cells treated with UCM or ACM for 24 hr. d , Representative images and quantification of 4.7 pN tension signal exerted by polyclonal CD4+ and e ,CD8+ T-cells treated with UCM or SCM for 24 hr. f, Schematic of a DNA-based tension probe presenting the SIINFEKL OVA-pMHC to antigen-specific OT-1 T-cells. g , Representative images and quantification of 4.7 pN tension signal exerted by OT-1 T-cells treated with UCM or ACM for 24 hr and h , UCM or SCM for 24 hr. Scale bars = 10 μm. Graphs represent means ± SD and data points represent fluorescence intensity measurements of individual cells. Statistics were calculated using a two-tailed student’s t-test. **** P < 0.0001, n = 2-3 independent animals.

Article Snippet: Murine CD4+ and CD8+ positive selection kits (# 130-117-043, 130-117-044), human CD3+ positive selection kit (# 130-050-101), and murine CD3+ and CD8+ negative selection kits (# 130-095-130, # 130-104-075) were purchased from Miltenyi Biotec.

Techniques: Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces

doi: 10.1101/2024.01.31.578101

Figure Lengend Snippet: a , Schematic illustration of ex vivo T cell activation experiments performed on mice fed with a control diet (CD, lean) or high fat diet (HFD, obese) for 3 months . b, Representative two-dimensional contour plots of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) (top), or perforin and granzyme B (GzmB) (bottom) for CD4+ and c, CD8+ T cells. d , Percent of T cells expressing TNF-⍺ (CD4+ ***P = 0.007, CD8+ ***P = 0.005) e , IFN-ɣ (***P = 0.0001, CD8+ P<0.0001) f , perforin (CD4+ ***P = 0.0002, CD8+ ***P = 0.0001), or g , granzyme B (****P<0.0001) after 72 h stimulation. Statistics were calculated using a one-way ANOVA with a Tukey’s multiple comparisons test. N = 3 mice per group. h , Representative images and i , quantification of 4.7 pN tension signal exerted by CD3+ T cells isolated from lean or obese mice (****P<0.0001). N = 3 mice from each group. j , Representative images and k , quantification of WAVE1 fluorescent intensity and l , cell spreading area of CD3+ T cells isolated from lean and obese mice (****P<0.0001). Statistics were calculated using an unpaired T test. N = 2 mice per group.

Article Snippet: Murine CD4+ and CD8+ positive selection kits (# 130-117-043, 130-117-044), human CD3+ positive selection kit (# 130-050-101), and murine CD3+ and CD8+ negative selection kits (# 130-095-130, # 130-104-075) were purchased from Miltenyi Biotec.

Techniques: Ex Vivo, Activation Assay, Control, Expressing, Isolation

Journal: bioRxiv

Article Title: The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces

doi: 10.1101/2024.01.31.578101

Figure Lengend Snippet: a , Schematic of the mechanism of action of blinatumomab. Blinatumomab is a bispecific T-cell engager (BiTE) that bridges CD3+ T-cells with CD19+ B-cells to induce cytolysis. b , Schematic of the blinatumomab DNA-based tension probe design (“blina-probe”). Blinatumomab is added to CD19-presenting tension probes to bridge CD3+ T-cells with CD19-presenting probes. c , Representative images and d , quantification of locked 4.7 pN tension signal of donor T-cells on blina-probes after a 24 hr treatment with UCM, SCM, or ACM. Circles (●) represent cells from donor 1, squares (▪) represent cells from donor 2, triangles (▴) represent cells from donor 3. Scale bar = 10 μm. Graphs represent means ± SD and data points represent fluorescence intensity measurements of individual cells. Statistics were calculated using a one-way ANOVA with a Tukey’s multiple comparisons test. **P < 0.0091, ****P < 0.0001, n = 3 independent donors.

Article Snippet: Murine CD4+ and CD8+ positive selection kits (# 130-117-043, 130-117-044), human CD3+ positive selection kit (# 130-050-101), and murine CD3+ and CD8+ negative selection kits (# 130-095-130, # 130-104-075) were purchased from Miltenyi Biotec.

Techniques: Fluorescence