lineage cell depletion kit  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: Stem cell factor 248 shapes ILC2 transcriptional programs and promotes mucosal inflammation in allergic asthma

doi: 10.3389/fimmu.2026.1843080

Figure Lengend Snippet: SCF248 expression impacts ILCp-ILC2 lineage specification in vitro . Bone marrow ILCp were sorted from naive Balb/c male mice. (A) ILCp sorting strategy. (B) SCF248 proinflammatory isoform was upregulated in OP9DL1 cells treated with rmIL-13 (10ng/mL for 16h). Gene expression analysis by qPCR was performed on ILCp cells differentiated in vitro for 72 h on OP9-DL1 cells or OP9-DL1 cells treated with IL-13. Cells were washed prior to co-culture. (C-I) Inhibitor of DNA binding 2 ( ID2 ), Gata3 , CD90 , Icos , c-Kit , Il4 , Il13 . (J) flow cytometry analysis of differentiated ILC2. Data are presented as mean ± SEM. Experiments were performed twice with n = 3–4 replicates per group. Statistical significance was determined using ordinary two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001.

Article Snippet: Next, we enriched our progenitor population by magnetically isolating Lineage + cells using a Direct Lineage Cell Depletion Kit and following the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Expressing, In Vitro, Gene Expression, Co-Culture Assay, Binding Assay, Flow Cytometry

Journal: Aging Cell

Article Title: Clonal Analyses Reveal the Impact of Hematopoietic Stem and Progenitor Cell Aging on T Cell Development

doi: 10.1111/acel.70615

Figure Lengend Snippet: Bulk young and aged hematopoietic stem and progenitor cells (HSPCs) produce similar cell output in vitro but young HSPCs develop more rapidly. (a) Mean cell numbers from thymi harvested from young (7–8 weeks old) and aged (18–24 months old) male and female C57BL/6J mice. Each dot represents an individual experiment with average of pooled data from 2 to 6 mice. Error bar denotes ± SD ( n = 54 mice, ordinary one‐way ANOVA). (b) Schematic of the mouse ATO system and key stages of T cell development. (c) Mean cell numbers at weeks 1, 3, and 6 of ATO initiated from bulk LSKs (Lineage‐Sca1+kit+) from young (7–8 weeks old) and aged (18.5–24 months old) male and female C57BL/6J mice. Each dot represents an average of pooled data from 12 ATOs. Error bar denotes ± SD ( n = 144 ATOs total from two independent experiments, ordinary two‐way ANOVA). (d) Frequencies of subsets of double negative (DN) cells (TCRβ‐CD3‐CD4‐CD8‐) at week 1 of ATO initiated from bulk LSKs isolated from young and aged male and female C57BL/6J mice, shown as a percentage of total DN cells. Each dot represents two pooled ATOs. Error bar denotes ± SD ( n = 48 ATOs total from two independent experiments, ordinary two‐way ANOVA). (e) Frequencies of cell subsets at week 3 of ATO initiated from bulk LSKs from young and aged male and female C57BL/6J mice. Frequencies of DN cells, immature single‐positive CD8+ (ISP8) cells (TCRβ‐CD3‐CD8+CD4‐), and double‐positive (DP) cells (TCRβ‐CD3‐CD8+CD4+) are shown as a percentage of total live CD45+Lin‐ cells. Each dot represents two pooled ATOs. Error bar denotes ± SD ( n = 48 ATOs total from two independent experiments, ordinary two‐way ANOVA). (f) Mean cell numbers at weeks 1, 3, and 6 of ATO initiated from bulk HSCs (LSK CD150+CD48‐) from young (7–8 weeks old) and aged (18.5–24 months old) male and female C57BL/6J mice. Each dot represents an average of pooled data from 12 ATOs. Error bar denotes ± SD ( n = 144 ATOs total from 2 independent experiments, ordinary two‐way ANOVA). (g) Frequencies of subsets of DN cells at week 3 of ATO initiated from bulk HSCs from young and aged male and female C57BL/6J mice, shown as a percentage of total DN cells. Each dot represents two pooled ATOs. Error bar denotes ± SD ( n = 48 ATOs total from two independent experiments, ordinary two‐way ANOVA). (h) Frequencies of cell subsets at week 6 of ATO initiated from bulk HSCs from young and aged male and female C57BL/6J mice. Frequencies of DN cells, ISP8 cells, and DP cells are shown as a percentage of total live CD45+Lin‐ cells. Each dot represents two pooled ATOs. Error bar denotes ± SD ( n = 48 ATOs total from two independent experiments, ordinary two‐way ANOVA). AF, aged female; AM, aged male; YF, young female; YM, young male. For all statistical analyses, only significant values are shown. A p ‐value of < 0.05 was deemed significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: Fresh bone marrow cells were enriched for HSPCs by negative cell selection of Lin‐ cells using magnetic cell sorting (MACS) with the mouse Direct Lineage Cell Depletion kit (Miltenyi Biotec, Cat #130‐110‐470).

Techniques: In Vitro, Isolation

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of a hematopoietic stem cell-delivered cell-penetrating frataxin in Friedreich’s ataxia models

doi: 10.1016/j.xcrm.2026.102803

Figure Lengend Snippet: LV-FXN gene therapy does not affect the engraftment and lineage commitment of HSPCs and deposits FXN protein in FRDA-relevant tissues (A) Overview of the transplantation experiment. Lineage-negative cells isolated from LY5.1 mice were transduced with LV-FXN at a multiplicity of infection (MOI) of 20 and transplanted into lethally irradiated LY5.2 recipient mice. Three months after transplantation, hematopoietic organs were analyzed by FACS, and frataxin protein levels were measured in the spleen, brain, heart, muscle, liver, and kidney by mass spectrometry. (B–D) Percentage of CD45.1 (donor-derived) and CD45.2 (recipient-derived) cells in peripheral blood, bone marrow (BM), and spleen of mice transplanted with mock-untransduced ( n = 3) or LV-FXN-transduced cells ( n = 4) (upper), along with the lineage composition within the CD45.1 and CD45.2 compartments (lower). (E) Levels of human mature frataxin (ng per mg of total protein; mean ± SD) in the spleen of mice transplanted with mock-untransduced cells (mouse #304) or LV-FXN-transduced cells (mice #306, #307, and #310). (F) Total frataxin levels (ng per mg of total protein; mean ± SD) in the indicated organs of mice transplanted with mock-transduced cells (mouse #304) or LV-FXN- transduced cells with >1 vector copy number (VCN) (mice #307 and #310). ND = not determined.

Article Snippet: Cells were pelleted and enriched for Ter119 - , Gr1, Mac1 - , B220 - , CD4 − , CD8 − , IL7R − and Sca + , cKit + cells by magnetic cell sorting using mouse Lineage Cell Depletion Kit (Miltenyi Biotech) according to the manufacturer’s protocol.

Techniques: Transplantation Assay, Isolation, Transduction, Infection, Irradiation, Mass Spectrometry, Derivative Assay, Plasmid Preparation

Journal: Blood Science

Article Title: Sequential in vivo CRISPR screens identify the clonal dominance of Nf1 loss in long-term hematopoiesis

doi: 10.1097/BS9.0000000000000294

Figure Lengend Snippet: Validation of the lentiviral screening system and identification of Nf1 enrichment. (A) Schematic representation of the quality control screening experiment using wild-type HSPCs to assess sgRNA library recovery and frequency distribution after long-term hematopoietic reconstitution. (B) Representative histograms of Ametrine fluorescence intensity (BV510) showing stable transduction efficiency in BM HSPC subsets 5 mo post-transplantation. (C) Correlation plot of sgRNA frequencies between different sorted hematopoietic subsets, demonstrating high reproducibility of the library distribution (*** p < .001). (D) Schematic representation of the primary transcription factor CRISPR screen performed using Cas9-expressing HSPCs. (E) Representative histograms displaying the transduction efficiency of the lentiviral TF library in different subsets of input c-Kit–enriched donor Cas9-expressing HSPCs. (F) Representative histograms showing the stable maintenance of lentiviral library expression within different subsets in the bone marrow 5 mo post-transplantation. (G) MAGeCK RRA score analysis highlighting significantly enriched genes. The RRA-positive score signified the statistical significance of a gene’s enrichment and a value closer to zero denoted stronger evidence. Nf1 was identified as the top hit. BM = bone marrow, CMP = common myeloid progenitor, GMP = granulocyte/monocyte progenitor, HSPC = hematopoietic stem and progenitor cell, MEP = megakaryocyte/erythroid progenitor, TF = transcription factor, WT = wildtype.

Article Snippet: Primary Cas9-expressing HSPCs (3–6 × 10 6 cells) were isolated from individual Cas9 transgenic mice using either a Lineage Cell Depletion Kit (Miltenyi, 130-110-470, Bergisch Gladbach, Germany) or CD117 MicroBeads (Miltenyi, 130-097-146), according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Control, Fluorescence, Transduction, Transplantation Assay, CRISPR, Expressing