hcd45 fitc  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a Schematic of wildtype and large deletion cassettes used to establish the LoD of large deletions, and representation of clonal expansion of cells harbouring a large deletion (orange) mediated genotoxic aberration amongst wildtype cells (blue). Dark and light grey bar indicates CCR5 and reference sequences, respectively. Red bar indicates flag sequence used to fuse assay and reference sequences. b Correlation of observed against the expected percentage of large deletions. Vertical black dotted line represents the limit of detection. Solid and dotted red line represents line of regression with 95% confidence interval, respectively. R 2 = 0.997. Calculated with linear regression analysis in GraphPad. Data shown as mean ± s.d. of n = 4 technical replicates. c CLEAR-time dPCR summaries of Cas9-edited HSPCs targeting various genes at different timepoints. The BTK edited HSPCs were also transduced with AAV6 encoding GFP. All editing was normalised against unedited mock electroporated HSPCs. Data are shown as mean ± s.d. d CLEAR-time dPCR summary of SH2D1A edited T cells with Cas9, Cas12, and TALENs at 3 days post-editing. Data are shown as mean ± s.d. e CLEAR-time dPCR summary of on-target CCR5 edited with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. f DSB and indels quantification at three known off-targets targeting CCR5 with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. g CLEAR-time dPCR on XIAP edited HSPCs pre-transplant and 16 weeks post-transplant. Data are shown as mean ± s.d. n = 4 mice for each treatment group. h Integration frequency in 16-week post-transplant XIAP edited and AAV-transduced hCD45 cells by dPCR and flow cytometry. Flow cytometry data represent n = 1 per mouse, dPCR data shown as mean ± s.d. of n = 3 technical replicates. i CLEAR-time dPCR normalised ICE analysis of pre-transplant XIAP edited HSPCs and post-transplant XIAP edited hCD45 cells. All data represents n = 3 technical replicates unless stated otherwise. d – f Two-way ANOVA with Tukey post-hoc test. n.s.= no statistical significance, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human CD45-positive cells were positively enriched using hCD45 microbeads (Miltenyi Biotec) and MS columns (Miltenyi Biotec) following manufacturer instructions.

Techniques: Sequencing, Transduction, TALENs, Flow Cytometry

Journal: bioRxiv

Article Title: Profiling histone post-translational modifications to identify signatures of epigenetic drug response in T-cell acute lymphoblastic leukemia

doi: 10.1101/2025.09.01.673463

Figure Lengend Snippet: (A) NSG mice were engrafted with malignant primary cells derived from the bone marrow of 10 T-ALL patients for tumor expansion. Upon disease establishment, mice were sacrificed, and leukemic cells were harvested from the spleen. To mimic biological replicates, six mice were injected per patient. The percentage of human CD45+ cells (hCD45%) was measured by flow cytometry, with only samples exceeding 85% used for downstream analysis. Global hPTM profiling and DNA methylation sequencing were performed for all 10 PDX models. In parallel, PDX cells were treated ex vivo with a panel of eight epidrugs to assess drug sensitivity. Spearman correlation analysis was then conducted to explore the relationship between hPTM levels and drug response. Figure created with Biorender.com . (B) Clustered and annotated heatmap displaying the normalized and summarized single hPTM levels across the 10 PDX models. (C) Volcano plot illustrating the differential analysis of hPTMs between CIMPlow and CIMPhigh PDX models.

Article Snippet: Lymphoblast engraftment and leukemic burden was monitored by flow cytometric analysis on peripheral blood, using human CD45+ (hCD45) staining (CD45-FITC antibody; Miltenyi Biotec, Bergish Gladbag, Germany).

Techniques: Derivative Assay, Injection, Flow Cytometry, DNA Methylation Assay, Sequencing, Ex Vivo