direct lineage cell depletion kit  (Miltenyi Biotec)

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