magnetic selection  (Miltenyi Biotec)

Journal: Cell Reports Medicine

Article Title: ABO: A 3D stroma-supported culture platform enabling full human B-lymphopoiesis for disease modeling and gene therapy development

doi: 10.1016/j.xcrm.2026.102879

Figure Lengend Snippet: B cell development in ABOs is not driven by expansion of pre-existing B-lineage-committed progenitor cells (A) Flow cytometric analysis of CD34, c-KIT (CD117), CD38, and CD10 expression on HSPCs before (top) and after depletion of Lin + CD10 + cells (bottom) by MACS, visualized with opt-SNE. (B and C) Flow cytometric analysis comparing the frequencies of CD33 + myeloid-committed (B) and CD10 + CD38 + lymphoid-committed cells (C) in day 35 ABOs using bulk or Lin + CD10 + -depleted CD34 + HSPCs. (D) Quantification of CD33 + and CD10 + CD38 + cells in day 35 ABOs. (E) Flow cytometric analysis comparing IgM + IgD − (immature) and IgM + IgD + (transitional) B cell frequencies in day 35 ABOs using bulk or Lin + CD10 + -depleted CD34 + HSPCs. (F) Quantification of IgM + and IgM + IgD + B cell frequencies in day 35 ABOs. (G) Distribution of HSPCs along the B cell developmental trajectory in day 35 ABOs. (D, F, G) Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test (ns; non-significant). (A–G) Data represent n = 3 individual donors in independent cultures. See also .

Article Snippet: Leukocytes from UCB and mPB were first purified using the Erythrocyte Sedimentation Kit II (Miltenyi), followed by CD34 + cell enrichment with the CD34 MicroBead Kit UltraPure (Miltenyi) per manufacturer’s instructions.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Harnessing human immune system models to validate NADPH oxidase 1 inhibition as treatment for hepatocellular carcinoma

doi: 10.3389/fphar.2026.1808515

Figure Lengend Snippet: NOX1i in Myeloid-HIS-HCC mice reduces monocyte and tumor-associated macrophage infiltration. (A) Schematic overview of the experimental set-up. Male NSG-SGM3 mice (n = 11) were sublethally irradiated and the immune system was partly reconstituted using human cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs). Four weeks post humanization, orthotopic human HCC was induced and mice were treated with 50 µM ML171 (NOX1i; n = 6) or vehicle (n = 5), twice per week for 3 weeks. (B) Human-to-murine immune cell (CD45) chimerism in the indicated tissues. (C) Human engraftment based on the number of human CD45 cells. Data are represented as violin plots showing median and quartiles. (D,E) Human immune cell subsets (D) and monocyte subsets (E) in indicated tissues represented as percentage of human CD45 cells. (F) Number of human macrophages and monocytes in the indicated tissues. (G) Representative immunohistochemistry images of infiltrated human monocytes/macrophages in liver tissue of HCC-bearing humanized mice. Sections were stained for human CD14 (brown), or matched IgG control. Scale bars: 100 µm. (B,D,E) . Data are shown as mean (SD). (C–E) Adjusted p-values were calculated using multiple unpaired t-test with Holm-Šídák correction (D,E) , or p-values were calculated using unpaired t-test with Welch’s correction or unpaired Mann-Whitney U test based on normal distribution (C,E) . *p < 0.05. HCC: hepatocellular carcinoma, NOX1i: NOX1 inhibition, huCD45: human CD45, muCD45: murine CD45, NK = natural killer, DCs: dendritic cells.

Article Snippet: Freshly isolated CB-derived PBMCs, isolated from CB as described for buffy coat, were enriched for HSPCs based on positive immunomagnetic selection of CD34 + expressing cells using the human CD34 MicroBead Kit UltraPure (Miltenyi Biotec, #130–100-453) according to manufacturer’s guidelines.

Techniques: Irradiation, Derivative Assay, Immunohistochemistry, Staining, Control, MANN-WHITNEY, Inhibition

Journal: Nucleic Acids Research

Article Title: ZEB1 drives terminal erythroid maturation by controlling the GATA2–KLF1 regulatory switch

doi: 10.1093/nar/gkag613

Figure Lengend Snippet: ZEB1 is required for optimal TED in primary human cells. ( A ) Experimental scheme of ZEB1 KD in primary human CD34 + HSPCs. ( B ) ZEB1 mRNA levels measured at day 3 of differentiation. ( C ) GPA levels at day 3 of erythroid differentiation. Percentages of GPA + cells are indicated. ( D ) Cell growth rates at days 3, 7, and 10. ( E ) Proportion of erythroid progenitors, pro-erythroblasts (Pro-E), and basophilic erythroblasts (Baso) at day 3 of erythroid differentiation. ( F ) mRNA levels of GATA2 and KLF1 in ZEB1 KD cells (day 3). ( G ) Enucleation rates measured by Syto-60 staining at day 10 of differentiation. Data are mean ± SEM of n = 3–5 independent biological experiments, from three independent blood donors, * P < .05, ** P < .01, *** P < .001, **** P < .0001 (Student’s t -test).

Article Snippet: CD34 + were isolated by positive selection using the CD34 MicroBead Kit (MACS Miltenyi) according to the manufacturers’ recommendation and cultured following a human ex vivo differentiation protocol as previously described [ ].

Techniques: Staining

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s can be generated from blood progenitors. CD34-positive cells isolated from blood were differentiated into cDC1s and cDC2s in a 17-day protocol in which progenitors initially expand and afterward undergo skewing towards cDC1 and cDC2 differentiation. (A) Schematic representation of the 17-day protocol for DC generation. (B) Bar graph showing the percentage of cDC1s and cDC2s ± SEM at the end of the culture on day 17 ( n = 10). (C) Representative dot plot showing the presence of cDC1s and cDC2s based on the expression of CD141/CLEC9A and CD1c, respectively, after gating on viable single cells. (D) Violin plot showing the absolute number of cDC1s and cDC2s obtained with the current protocol, depending on the initial amount of cultured CD34-positive cells. Each dot represents the value of expanded cDC1s and cDC2s obtained from one independent donor ( n = 12). (E) Bar graphs show the percentage of positive DCs ± SEM for the given marker ( n = 3). CD34-derived cDC1s and cDC2s were characterized by flow cytometry to determine their lineage marker expression.

Article Snippet: CD34 + cells were isolated with the MACS CD34 Microbead kit Ultrapure (130-100-453, Miltenyi Biotec).

Techniques: Derivative Assay, Generated, Isolation, Expressing, Cell Culture, Marker, Flow Cytometry

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s are endocytic and exhibit antigen-presenting capacity. (A) Dot plots showing the percentage of cDC1s and cDC2s positive for CTV signal, indicative of CTV-labeled tumor cell uptake. (B) The scatter dot plot shows the frequency of tumor cell uptake positive DCs (identified as the percentage of CTV positive cDC1s and cDC2s) (mean ± SEM). (C) Representative histograms showing the percentage of cDC1s and cDC2s positive for Alexa647, indicative of active Dextran-Alexa647 uptake. (D) Scatter dot plot displaying the active dextran uptake (mean ± SEM). (E) Representative dot plots showing the frequency of proliferating autologous CFSE-labeled pan T-cells (indicated by the low CFSE expression) upon coculture with either untreated, TT-stimulated, or CMV-stimulated cDC1s for 6 d. (F) Scatter dot displaying the mean ± SEM of the percentage of autologous proliferating T-cells upon coculture with cDC2s (blue) and cDC1s (red) for 6 d under the indicated antigenic stimuli. (G) Scatter dot showing the mean ± SEM of the raw concentration of IFNγ and IL-2 detected by Luminex assay on supernatants collected after 6 d of cDC1 and cDC2 coculture with autologous T-cells together with the indicated antigenic stimuli. Statistical significance during uptake assays was calculated with either an unpaired t-test or a Mann–Whitney test. Statistical significance across antigen-specific assays was determined with Kruskal–Wallis test for the multiple comparison. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: CD34 + cells were isolated with the MACS CD34 Microbead kit Ultrapure (130-100-453, Miltenyi Biotec).

Techniques: Derivative Assay, Labeling, Expressing, Concentration Assay, Luminex, MANN-WHITNEY, Comparison

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s induce tumor-derived antigen-specific CD8 T-cell clones. To address the capacity of these DCs to prime the induction of a T-cell clone specific against the tumor antigen MART1, matured CD34-derived pan-DCs (combined cDC1s and cDC2s) loaded with the MART1-derived peptide were cultured with autologous naive CD8 T-cells. (A) Schematic representation of the experimental layout. (B) Representative dot plot showing the frequency of MART1-positive CD8 T-cells after the induction protocol with CD34-derived pan-DCs, as indicated by the abundance of dextramer-PE positive CD8 T-cell. (C) Before–after dot plots showing the frequency of MART1-specific dextramer-stained CD8 T-cells for each donor compared to a negative control dextramer. Each dot represents an individual donor ( n = 4). (D) Schematic representation of the rechallenge experimental layout. To address the responsiveness of induced MART1-specific T-cells to a secondary antigen rechallenge, MART1-loaded autologous moDCs were cocultured overnight with the earlier primed CD8 T-cell pool containing the MART1-specific CD8 T-cells. (E) Representative dot plots showing the expression levels of CD137 and CD25 on CD8 T-cells cocultured with either unloaded or MART1-loaded moDCs. (F) Before–after dot plots showing the frequency of positive CD8 T-cells for the simultaneous expression of CD25 and CD137. (G) Before–after dot plots showing the frequency of positive CD8 T-cells for the activation marker CD25, CD137, CD69, CD107a, and the intracellular cytokines IFNγ, IL-2, and TNFα after coculture with either unloaded or MART1-loaded moDCs. Each dot represents an individual donor ( n = 4). Statistical significance was calculated using either a paired t-test or a Wilcoxon test. P- values are numerically depicted in the graph.

Article Snippet: CD34 + cells were isolated with the MACS CD34 Microbead kit Ultrapure (130-100-453, Miltenyi Biotec).

Techniques: Derivative Assay, Clone Assay, Cell Culture, Staining, Negative Control, Expressing, Activation Assay, Marker