clone 1d3  (Bio X Cell)

Journal: iScience

Article Title: The transcription factor IRF4 regulates the homeostasis and function of intestinal ILC3s

doi: 10.1016/j.isci.2025.112800

Figure Lengend Snippet: IRF4 deficiency compromises MHC class Ⅱ expression and further restrains ILC-mediated apoptosis of effector CD4 + T cells both in vitro and in vivo (A) Violin plots visualizing the expression of MHC-class-Ⅱ-related signature genes. (B) FACS analysis and MFI of MHC class Ⅱ expression in ILC3s isolated from Irf4 f/f and Irf4 f/f Rorc cre mice ( n = 6). ILC3 subsets were gated as Lin − RORγt + and then CCR6 + NKp46 − , CCR6 − NKp46 + , or CCR6 − NKp46 − . The lineage cocktail included TCRγδ, CD3ε, CD19, CD5, CD11c, Gr-1, and Ter119. (C and D) Activated OT-Ⅱ CD4 + T cells were cultured ex vivo with purified ILC3s from the siLP of Irf4 f/f and Irf4 f/f Rorc cre mice in the presence or absence of Ova peptide or the anti-MHC class Ⅱ neutralizing antibody. (C) Quantification of OT-Ⅱ T cells recovery (%). (D) Quantification of Annexin-V + OT-Ⅱ T cells. (E and F) Naive CD4-positive T cells (gating as CD4 + CD25 − CD62L hi CD44 lo cells) were sorted from OT-Ⅱ mice and pre-activated overnight. After pre-activation, CD4 positive T cells were transplanted into recipient Irf4 f/f and Irf4 f/f Rorc cre mice along with OVA peptide administration every 2 days following transfer. Nine days later, mice were sacrificed for further analysis of OT-Ⅱ CD4 + T cells (gating as CD8 − CD4 + TCRβ + Vβ5 + ) transferred in the spleen, mLN, siLPL, and cLPL of recipient mice. (G) One hundred thousand intestinal ILC3s (Lin − CD127 + CD27 − KLRG1 − ) were sorted from Irf4 f/f or Irf4 f/f Rorc cre mice and transferred with 500,000 activated OT-ⅡCD4 + T cells into NCG mice, flowing by OVA peptide i.p. every 2 days. Nine days later, survived OT-ⅡCD4 + T cells were quantified. ( n = 2 APC, n = 5 transferred group). Bar graphs are presented as mean ± SEM. A two-tailed Student’s t test was performed for comparisons. The data are representative of at least three independent experiments (B–G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also .

Article Snippet: Biotin CD19 (clone 1D3) , eBioscience , Cat# 13-0193-86; RRID: AB_657655.

Techniques: Expressing, In Vitro, In Vivo, Isolation, Cell Culture, Ex Vivo, Purification, Activation Assay, Two Tailed Test

Journal: Leukemia

Article Title: Trajectories from single-cells to PAX5-driven leukemia reveal PAX5-MYC interplay in vivo

doi: 10.1038/s41375-025-02626-2

Figure Lengend Snippet: A Percentage of BP-1 + bone marrow B-cells (B220 + CD19 + ) from Pax5 ± vs. wild-type (WT) mice. (Left) Displayed are individual values (with mean and SD) of 14 Pax5 ± vs. 14 WT mice ages 11-38 weeks. (Right) Representative flow cytometry plots. An unpaired two-tailed Student’s t-test was used to calculate significance and the respective p -value is indicated. *** p ≤ 0.001. B Schematic of the employed sorting and single-cell RNA-Sequencing strategy. C Single-cell clusters of different WT precursor B-cell subsets analyzed with the workflow depicted in B). The transcriptome similarity is visualized based on UMAP. Pool of n = 3 WT mice, 17 weeks of age. D Predicted cell-cycle state in clusters from C). E Precursor B-cells of WT mice display specific gene expression patterns depending on their differentiation state. The scaled gene expression level for classical marker genes ( Igll1 , Vpreb1 and Il2ra ) is shown in color on the UMAP. F Expressed immunoglobulin chain status of WT precursor B-cell clusters displayed as in C). Igh immunoglobulin heavy chain, Igk immunoglobulin kappa light chain, Igl immunoglobulin lambda light chain. True chain expression, False no chain expression, No data no chain detected.

Article Snippet: Panel 1 included anti-Human/Mouse CD45R (B220) FITC (clone RA3-6B2, 1:200), anti-Mouse CD19 eFluor 450 (clone eBio1D3 (1D3), 1:400), anti-Mouse IgM APC-eFluor 780 (clone II/41, 1:100), anti-Mouse CD117 APC (clone 2B8, 1:800), anti-Mouse CD25 PE-Cyanine 7 (clone PC61.5, 1:400), anti-Mouse CD3e PE (clone eBio500A2 (500A2), 1:100) - all from eBioscience (Thermo Fisher Scientific).

Techniques: Flow Cytometry, Two Tailed Test, RNA Sequencing, Gene Expression, Marker, Expressing

Journal: Leukemia

Article Title: Trajectories from single-cells to PAX5-driven leukemia reveal PAX5-MYC interplay in vivo

doi: 10.1038/s41375-025-02626-2

Figure Lengend Snippet: A Upper: Schematic of B-cell differentiation in the bone marrow. SL surrogate light chain, µH heavy chain. Lower: Population frequencies among the different differentiation stages (individual values with mean and SD). Compared are wild-type (WT) mice ( n = 3, 11 weeks) and Pax5 ± littermates ( n = 3, 11 weeks). Representative flow cytometry plots displaying the pre-BII population (B220 + CD19 + IgM − IgD − c-KIT − CD25 + ) for WT and Pax5 ± mice are displayed on the right. B Mean fluorescence intensity (MFI) of CD19 in pro-B cells of Pax5 ± mice compared to WT littermates. C Analogous to B) for CD25 MFI in pre-BII cells. D Percentage of IL7-Receptor (IL7-R, CD127) positive cells among pre-BII cells of Pax5 ± mice compared to their WT littermates. E qRT-PCR analysis showing Pax5 gene expression in B220-enriched bone marrow cells of 14 weeks old Pax5 ± mice compared to WT animals ( n = 3). F Frequencies of pro-B, pre-BI and pre-BII cells within the (B220 + CD19 + IgM − IgD − ) parental population in WT vs. Pax5 ± mice across different age cohorts (6 weeks, WT n = 4, Pax5 ± n = 5; 11 weeks, WT n = 3, Pax5 ± n = 3; 25–26 weeks, WT n = 4, Pax5 ± n = 3; 38 weeks, WT n = 4, Pax5 ± n = 5; 65–72 weeks, WT n = 3, Pax5 ± n = 3). w weeks. G Population frequencies among the different differentiation stages. Compared are WT mice ( n = 3, 2 weeks) and Pax5 ± littermates ( n = 3, 2 weeks). H Percentage of BP-1 + bone marrow B-cells (B220 + CD19 + ) from 2 weeks old Pax5 ± vs. WT mice ( n = 3 per group). I qRT-PCR analysis showing Pax5 gene expression in B220-enriched bone marrow cells of 2 weeks old Pax5 ± compared to WT animals ( n = 3 per group). Displayed are individual values with mean and SD. An unpaired two-tailed Student’s t-test was performed for the statistical analysis. Respective p -values are indicated. ns not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Panel 1 included anti-Human/Mouse CD45R (B220) FITC (clone RA3-6B2, 1:200), anti-Mouse CD19 eFluor 450 (clone eBio1D3 (1D3), 1:400), anti-Mouse IgM APC-eFluor 780 (clone II/41, 1:100), anti-Mouse CD117 APC (clone 2B8, 1:800), anti-Mouse CD25 PE-Cyanine 7 (clone PC61.5, 1:400), anti-Mouse CD3e PE (clone eBio500A2 (500A2), 1:100) - all from eBioscience (Thermo Fisher Scientific).

Techniques: Cell Differentiation, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Leukemia

Article Title: Trajectories from single-cells to PAX5-driven leukemia reveal PAX5-MYC interplay in vivo

doi: 10.1038/s41375-025-02626-2

Figure Lengend Snippet: A Single-cell (sc) RNA-Sequencing clusters of sorted pre-BII cells from n = 4 wild-type (WT) and n = 4 Pax5 ± mice ages 10-11 weeks (left) with annotated cell-cycle states (right). B : Top gene expression is shown for each cluster as a heatmap. C Cluster specific proportion of pre-BII cells from WT vs. Pax5 ± mice. D Proportion heatmaps showing the cell distribution of pre-BII cells among the different clusters from Pax5 ± and WT mice. The lower left clusters 4 and 6 (see Fig. 4A) correspond to lambda light chain recombination status in the V(D)J data (refer to Supplementary Fig. ). E V(D)J-recombination analysis showing annotation of immunoglobulin lambda light chain ( Igl ) expression. True chain expression, False no chain expression, No data no chain detected. F Flow cytometry analysis showing the percentage of kappa and lambda light chain rearranged B-cells among B220 + CD19 + cells in the peripheral blood of Pax5 ± ( n = 7) vs. WT ( n = 7) mice ages 11–16 weeks (individual values with mean and SD). Significances (indicated) were calculated using an unpaired two-tailed Student’s t-test. *** p ≤ 0.001, **** p ≤ 0.0001. G Pedigrees of two families harboring PAX5 germline variants (p.G183S and p.G183R, respectively). Epstein Bar Virus (EBV) transformed lymphoblastoid cell lines (LCL) were available from all individuals shaded in grey. BCP-ALL B-cell precursor acute lymphoblastic leukemia. H Flow cytometry analysis of IGK (immunoglobulin kappa light chain) vs. IGL surface expression levels on EBV LCLs depicted in G ). PAX5 p.G183S n = 3, PAX5 p.G183R n = 4, PAX5 WT n = 2.

Article Snippet: Panel 1 included anti-Human/Mouse CD45R (B220) FITC (clone RA3-6B2, 1:200), anti-Mouse CD19 eFluor 450 (clone eBio1D3 (1D3), 1:400), anti-Mouse IgM APC-eFluor 780 (clone II/41, 1:100), anti-Mouse CD117 APC (clone 2B8, 1:800), anti-Mouse CD25 PE-Cyanine 7 (clone PC61.5, 1:400), anti-Mouse CD3e PE (clone eBio500A2 (500A2), 1:100) - all from eBioscience (Thermo Fisher Scientific).

Techniques: RNA Sequencing, Gene Expression, Expressing, Flow Cytometry, Two Tailed Test, Virus, Transformation Assay

Journal: Leukemia

Article Title: Trajectories from single-cells to PAX5-driven leukemia reveal PAX5-MYC interplay in vivo

doi: 10.1038/s41375-025-02626-2

Figure Lengend Snippet: A Schematic of transplantation strategy. B Flow cytometry analysis showing the percentage of engrafted donor CD45.2 + pre-BII cells in lethally irradiated recipients 72 h after transplantation in the bone marrow (upper) and the spleen (lower). Donor cells were a pool of n = 4, 11 weeks old either Pax5 ± or wild-type (WT) mice, each transplanted into n = 5 CD45.1 + lethally irradiated recipients. No support whole bone marrow cells (WBMCs) was used for this experimental setup. “Among B-cell lineage” refers to gating on scatter, singlets, viable, lineage and B220 + CD19 + . C Population frequencies of precursor B-cell subsets 72 h after in vitro cultivation of B220 + sorted cells from WBMCs of WT ( n = 4) vs. Pax5 ± ( n = 4) mice, ages 9-10 weeks. Displayed are individual values with mean and SD. An unpaired two-tailed Student’s t -test was performed for the statistical analysis. Respective p -values are indicated. ns not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Panel 1 included anti-Human/Mouse CD45R (B220) FITC (clone RA3-6B2, 1:200), anti-Mouse CD19 eFluor 450 (clone eBio1D3 (1D3), 1:400), anti-Mouse IgM APC-eFluor 780 (clone II/41, 1:100), anti-Mouse CD117 APC (clone 2B8, 1:800), anti-Mouse CD25 PE-Cyanine 7 (clone PC61.5, 1:400), anti-Mouse CD3e PE (clone eBio500A2 (500A2), 1:100) - all from eBioscience (Thermo Fisher Scientific).

Techniques: Transplantation Assay, Flow Cytometry, Irradiation, In Vitro, Two Tailed Test

Journal: Leukemia

Article Title: Trajectories from single-cells to PAX5-driven leukemia reveal PAX5-MYC interplay in vivo

doi: 10.1038/s41375-025-02626-2

Figure Lengend Snippet: A Representative flow cytometry plots showing a low percentage of a deregulated population (c-KIT + CD25 + ) in the bone marrow of a 38 weeks old Pax5 ± mouse (highlighted with arrow; termed pre-leukemia). This was accompanied with an enrichment of pre-pro B cells (B220 + CD19 − ). WT wild-type. B Single-cell RNA-Sequencing (scRNA-Seq) analysis including WT precursor B-cell subsets from Fig. and the pre-leukemic population identified in A). Pre-leukemic cells cluster closest to WT pro-B cells. C Gene expression profile of selected genes in pre-leukemic cells (pre-leuk) compared to WT B-cell differentiation stages extracted from the scRNA-Seq analysis as a dot plot heatmap. Cluster labels are displayed in B). D Transcription factor (TF) activities in pre-leukemic cells compared to WT B-cell subsets based on the SCENIC analysis. Important TF regulons that are involved in hematopoietic cell differentiation identity are highlighted. See also Supplementary Fig. showing the predicted activity of PAX5 and EBF1 regulons within our recorded scRNA-Seq data of different WT B-cell subsets on the UMAP. Imm-B Immature B-cells, Recirc-B Recirculating B-cells, c cycling. E Three most significantly enriched motif classes are shown from differentially accessible peaks comparing Pax5 ± pre-leukemic (pre-leukemia 5 and 7) and WT pre-BII cells (up/down: higher/lower accessibility in pre-leukemia, respectively, refer to Supplementary Table ). The dot size corresponds to the fraction of peaks carrying the motif. Color denotes -10log( p -value). F Histogram of ATAC-seq signal level at PAX5 binding sites comparing Pax5 ± pre-leukemic ( n = 2, pre-leukemia 5 and 7) and WT pre-BII ( n = 2) chromatin access. Peaks centered with PAX5 motif are shown ( n = 963 peaks, ± 1 kb from center, re-analysis of CUT & RUN data from pro-B and pre-B cells ).

Article Snippet: Panel 1 included anti-Human/Mouse CD45R (B220) FITC (clone RA3-6B2, 1:200), anti-Mouse CD19 eFluor 450 (clone eBio1D3 (1D3), 1:400), anti-Mouse IgM APC-eFluor 780 (clone II/41, 1:100), anti-Mouse CD117 APC (clone 2B8, 1:800), anti-Mouse CD25 PE-Cyanine 7 (clone PC61.5, 1:400), anti-Mouse CD3e PE (clone eBio500A2 (500A2), 1:100) - all from eBioscience (Thermo Fisher Scientific).

Techniques: Flow Cytometry, RNA Sequencing, Gene Expression, Cell Differentiation, Activity Assay, Binding Assay

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet:

Article Snippet: CD19 − Pacific Blue (Clone: 1D3) , ThermoFisher Scientific , 48-0193-82; RRID:AB_2734905.

Techniques: Purification, Recombinant, cDNA Synthesis, Staining, RNA Sequencing, Cloning, Software