cd34 selection beads kit  (Miltenyi Biotec)

Journal: Cell reports

Article Title: Differentiation latency and dormancy signatures define fetal liver hematopoietic stem cells at single-cell resolution

doi: 10.1016/j.celrep.2025.116289

Figure Lengend Snippet: (A) Sorting strategy for SE hi FL-HSCs from freshly isolated E13.5 FL used for scRNA-seq. (B) Unsupervised clustering of E13.5 SE hi FL-HSC scRNA-seq data in UMAP. (C) Heatmap of gene-set scores by cluster for genes associated with adult HSC dormancy, serial-engrafting HSCs, diapause, , chemokine signaling (WP_CHEMOKINE_SIGNALING_PATHWAY), and a common stem-cell dormancy state associated with lipid metabolism, or genes associated with activated HSC/MPP states including high output and multilineage signatures, , , Myc pathway activation (Hallmark Myc Target Genes V1, V2), and metabolic activity (WP_TCA_CYCLE, HALLMARK_OXIDATIVE_PHOSPHORYLATION, WP_PURINE_METABOLISM). (D) Gene-set expression heatmaps for dormant HSC signature genes and serial-engrafting HSC signature genes. , (E) Expression heatmaps for modules of co-regulated genes. Gene modules 1, 3, 4, and 7 are shown with representative genes identified in each module.

Article Snippet: After centrifugation, the pellet was resuspended in 4 mL MACs buffer (MACS BSA Stock Solution Miltenyi Biotec 130–091-376, in autoMACs Rinsing Solution, Miltenyi Biotec 130–091-222) for CD34 enrichment using CD34 selection beads/kit (Miltenyi Biotech, 130–100-453) according to the manufacturer protocol.

Techniques: Isolation, Activation Assay, Activity Assay, Phospho-proteomics, Expressing

Journal: Nature Cancer

Article Title: Aberrant expression of SLAMF6 constitutes a targetable immune escape mechanism in acute myeloid leukemia

doi: 10.1038/s43018-025-01054-6

Figure Lengend Snippet: a , Schematic overview of the antibody screen to identify novel markers differentially expressed on CD34 + CD38 low cells from NBM and from participants with AML with TP53 mutations by flow cytometry. b , Most highly expressed cell surface markers on CD34 + CD38 low cells in AML ( n = 3 participants) compared to NBM ( n = 3 donors) samples. Previously identified AML markers are shown in bold. c , Expression of SLAMF6 on CD34 + CD38 low cells from representative samples from NBM and a participant with AML. d , SLAMF6 expression on the three AML cases and three NBM samples included in the antibody screen (two-sided t -test, P = 0.011). e , Expression of SLAMF6 on normal hematopoietic cell populations in peripheral blood from healthy donors (one representative donor of four). f , Expression of SLAMF6 in hematopoietic cell types based on scRNA-seq of mononuclear cells from five NBM donors and CD34 + cells from three donors. In the violin plot, dots represent individual donors, bars indicate median values and blue shading indicates density. g , SLAMF6 expression in NBM populations from an scRNA-seq dataset with ten healthy donors . In the violin plot, dots represent individual donors, bars indicate median values and blue shading indicates density.

Article Snippet: Treatment experiments with CD34 + cells from healthy donors were performed in 8–12-week-old female mice of the NSG mouse strain (Jackson Laboratory) transplanted with CD34 + cord blood cells isolated by Lymphoprep separation (GE Healthcare) and subsequent CD34 positive selection using magnetic beads (Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing

Journal: Nature Cancer

Article Title: Aberrant expression of SLAMF6 constitutes a targetable immune escape mechanism in acute myeloid leukemia

doi: 10.1038/s43018-025-01054-6

Figure Lengend Snippet: a , Percentage of AML samples expressing SLAMF6 on the CD33 + blast cell population ( n = 50 cases). b , Percentage of the CD34 + AML samples expressing SLAMF6 on the CD34 + CD38 low cell population ( n = 35 cases). Frequency of AML cases expressing SLAMF6, stratified by disease subtype as defined by the ICC 2022 classification ( c ), recurrent mutations ( d ), ELN 2022 risk group classification ( e ) and French–American–British classification ( f ). g , SLAMF6 expression on cases of relapsed/refractory AML. Dark blue, high SLAMF6 expression; light blue, intermediate SLAMF6 expression; gray, no SLAMF6 expression. h , SLAMF6 expression on myeloid leukemia cell lines by flow cytometry. Blue, staining with SLAMF6 antibody; gray, staining with isotype-matched control antibody. Correlation between the expression of SLAMF6 and that of CD200 ( i ), CD47 ( j ), CD84 ( k ), CD244 ( l ) and PDL1 ( m ) on primary AML samples, based on flow cytometry (Kruskal–Wallis test with Dunn’s post hoc test; n = 20 cases for CD200 and PDL1 and n = 39 cases for CD47, CD84 and CD244). Bars indicate median values. CEBPA , AML with in-frame bZIP CEBPA mutations; MECOM , AML with other MECOM rearrangements; NOS, AML not otherwise specified.

Article Snippet: Treatment experiments with CD34 + cells from healthy donors were performed in 8–12-week-old female mice of the NSG mouse strain (Jackson Laboratory) transplanted with CD34 + cord blood cells isolated by Lymphoprep separation (GE Healthcare) and subsequent CD34 positive selection using magnetic beads (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Nature Cancer

Article Title: Aberrant expression of SLAMF6 constitutes a targetable immune escape mechanism in acute myeloid leukemia

doi: 10.1038/s43018-025-01054-6

Figure Lengend Snippet: ( a ) Schematic overview of the humanized mouse model without human PBMCs. Five mice were treated with TNC-1 and four mice were treated with an isotype control antibody (all statistical testing performed with two-sided Mann-Whitney U test). ( b ) AML engraftment in bone marrow after treatment with TNC-1 following transplantation of HNT-34 AML cells without co-transplantation of human PBMCs, determined as the percentage of the total cell population constituted by human AML cells (p = 0.413). ( c ) AML engraftment in spleen (p = 0.413). ( d ) Weights of isolated spleens (p = 0.413). ( e ) T cell-mediated killing of HNT-34 cells in reponse to treatment with TNC-1 in 72 h co-culture experiments with primary T cells and SLAMF6 wildtype and knockout AML cells. Mean ± SEM of four T cell donors, normalized to SLAMF6 wildtype and isotype control (two-sided Mann-Whitney U test; p = 0.029 for WT, p = 0.886 for KO (A), p = 0.886 for KO (B)). ( f ) T cell-mediated killing of KG-1 cells in T cell co-culture assay. Mean ± SEM of four T cell donors, normalized to SLAMF6 wildtype and isotype control (two-sided Mann-Whitney U test; p = 0.029 for WT, p = 0.886 for KO (A), p = 0.886 for KO (B)). ( g ) Schematic overview of the mouse model with SLAMF6 knockout HNT-34 cells. Six mice were treated with TNC-1 and six mice were treated with an isotype control antibody (all statistical testing performed with two-sided Mann-Whitney U test). ( h ) AML engraftment in bone marrow after treatment with TNC-1 following transplantation of primary human T cells and SLAMF6 knockout HNT-34 cells, determined as the percentage of the total cell population constituted by human AML cells (p = 0.818). ( i ) AML engraftment in spleen (p = 0.589). ( j ) Weights of isolated spleens (p = 0.485). Bone marrow engraftment of ( k ) CD34 + cells (Wilcoxon signed-rank test; p = 0.063) and ( l ) CD34 + CD38 low cells (Wilcoxon signed-rank test; p = 0.625) in response to treatment with TNC-1 after transplantation of CD34 + cells from human umbilical cord blood. For each of five different cord blood donors, one mouse was treated with TNC-1 and one with an isotype control antibody. Connecting lines indicate mice engrafted with cells from the same donor.

Article Snippet: Treatment experiments with CD34 + cells from healthy donors were performed in 8–12-week-old female mice of the NSG mouse strain (Jackson Laboratory) transplanted with CD34 + cord blood cells isolated by Lymphoprep separation (GE Healthcare) and subsequent CD34 positive selection using magnetic beads (Miltenyi Biotec).

Techniques: Control, MANN-WHITNEY, Transplantation Assay, Isolation, Co-Culture Assay, Knock-Out, Co-culture Assay

Journal: Nature Cancer

Article Title: Aberrant expression of SLAMF6 constitutes a targetable immune escape mechanism in acute myeloid leukemia

doi: 10.1038/s43018-025-01054-6

Figure Lengend Snippet: Left, SLAMF6 is upregulated in 60% of AML cases but not expressed on healthy HSPCs. Middle, SLAMF6 mediates protection against T cell-mediated killing. Genetic knockout or blocking antibodies reverse this effect and unleash a potent antileukemia T cell response. Right, the SLAMF6-blocking antibody TNC-1 induces a strong and specific immune response that reduces leukemia burden in humanized mice while sparing healthy stem and progenitor cells.

Article Snippet: Treatment experiments with CD34 + cells from healthy donors were performed in 8–12-week-old female mice of the NSG mouse strain (Jackson Laboratory) transplanted with CD34 + cord blood cells isolated by Lymphoprep separation (GE Healthcare) and subsequent CD34 positive selection using magnetic beads (Miltenyi Biotec).

Techniques: Knock-Out, Blocking Assay

Journal: bioRxiv

Article Title: Polymer-based culture system enables expansion of human haematopoietic stem and progenitor cells while preserving ageing-associated transcriptional programs

doi: 10.1101/2025.09.10.675303

Figure Lengend Snippet: A) Experimental outline. Created with BioRender.com B) UMAP of predicted cell types from scRNA-seq - all samples combined. C) UMAP of predicted cell types by HSPC sample type/source (cord blood, bone marrow young, bone marrow old). D) Expression of stem cell signature genes CRHBP and MECOM in different HSPC sample types. E) Expression of myeloid signature genes MPO and LYZ . F) AUCell score for “old” signature. G) AUCell score for “young” signature. Significant differences were determined using the Wilcoxon rank-sum test with Benjamini–Hochberg correction. **** p . adj < 0.0001

Article Snippet: CD34 + cell selection: All samples were then enriched for CD34 + cells using a CD34 MicroBead Kit UltraPure and an AutoMACS machine (Miltenyi Biotec), according to the manufacturer’s protocols.

Techniques: Expressing

Journal: bioRxiv

Article Title: Polymer-based culture system enables expansion of human haematopoietic stem and progenitor cells while preserving ageing-associated transcriptional programs

doi: 10.1101/2025.09.10.675303

Figure Lengend Snippet: A) Experimental outline. Created with BioRender.com B) UMAP of predicted cell types from scRNA – all samples combined. C) UMAP of predicted cell types by HSPC sample type/source (cord blood, peripheral blood young, peripheral blood old). D) AUCell score for “old” signature. E) AUCell score for “young” signature. Significant differences were determined using the Wilcoxon rank-sum test with Benjamini– Hochberg correction. **** p . adj < 0.0001. F) % phenotypic haematopoietic stem and progenitor cells (pHSPCs) within the live fraction. Data are shown as mean +/-SD. Mann-Whitney test was performed. G) % phenotypic haematopoietic stem cells (pHSCs) within the CD34 + CD38-fraction. Data are shown as mean +/-SD. Mann-Whitney test was performed. H) % of EPCR+ cells within the HSC fraction from cells expanded for 30 days. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001.

Article Snippet: CD34 + cell selection: All samples were then enriched for CD34 + cells using a CD34 MicroBead Kit UltraPure and an AutoMACS machine (Miltenyi Biotec), according to the manufacturer’s protocols.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Generation of effector CD4+ T cells from Human iPSC

doi: 10.1101/2025.08.01.667959

Figure Lengend Snippet: ( A ) Schematic of iT differentiation to CD4+ cells. ( B ) Representative flow cytometry of key developmental timepoints and metrics of DP T cells. iPSCs were plated at day −2 and cultured for 12 days in HSPC differentiation conditions. The resultant CD34+ HSPCs were purified using magnetic beads. Day 12 HSPCs were moved to Notch-stimulatory lymphoid coating and cultured with StemCell Technologies lymphoid expansion media until day 26, where cells were assayed for CD7 expression. Day 26 cells were replated on fresh Notch stimulatory coating and cultured with StemCell Technologies lymphoid maturation media to day 40, where cells are assayed for TCR, CD3, CD4 and CD8 expression. (N=3) ( C ) Representative flow cytometry of D54 T cells, with parent gating in Figure S3A. Day 40 iPSC DP T cells were moved to Retronectin coating and treated with anti-CD3 antibody (OKT3, 5 ug/ml), half media changes were performed every 3 or 4 days. (N=3) ( D ) Representative flow cytometry of D68 T cells post expansion. Day 54 iCD4+ T cells were moved to fresh Retronectin coated wells and treated with media supplemented with anti-CD3/CD28 and 200 U/ml IL-2. (N=3) ( E ) Percentage of CD4 and CD8 positive T cells from iCD4+ T cell differentiation by flow cytometry at day 40, 54 and 68, N=3, analyzed by 2-way ANOVA followed by a Tukey’s multiple comparison test, *P < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001. ( F ) Representative flow cytometry of T cells from long-term culture in X-Vivo 15 media supplemented with 200 U/ml IL-2. N=3, parent gating for each plot as indicated. (G) Number of doublings of iCD4+ T cells after stimulation with anti-CD3/CD28 (N = 3).

Article Snippet: CD34+ progenitors are isolated from all floating cells by MACS separation using Miltenyi CD34+ selection kit and LS columns (Miltenyi).

Techniques: Flow Cytometry, Cell Culture, Purification, Magnetic Beads, Expressing, Cell Differentiation, Comparison