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Easily isolate highly purified and magnetic particle-free human CD45+ leukocytes from single-cell suspensions of primary human tissues and tumors (Catalog #100-0105), or tissues and tumor xenografts from humanized mice (Catalog #100-0107) samples, by immunomagnetic positive selection, with the EasySep™ Release Human CD45 Positive Selection Kit. Widely used in published research for more than 20 years, EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system.
In this EasySep™ positive selection procedure, desired cells are first labeled with antibody complexes recognizing CD45 and magnetic particles called EasySep™ Releasable RapidSpheres™. Unlike traditional magnetic particles, which stay bound to the target cells, these RapidSpheres™ have a releasable feature. After separation using an EasySep™ magnet, bound magnetic particles are removed from the EasySep™-isolated CD45+ cells using a release agent, and unwanted cells are targeted for depletion. The final isolated fraction contains highly purified CD45+ cells that are immediately ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction. Following cell isolation with this EasySep™ kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violet™ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands.
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including those for culture media, supplements, antibodies, and more.
Figure 1. EasySep™ Release Human CD45 Positive Selection Kit
Starting with a single-cell suspension of a human breast cancer tumor xenograft (MDA-MB-231) sample from an NRG-3GS humanized mouse, the CD45+ TIL purities of the start and final isolated fractions are 5.5% and 96.0%, respectively.
NOTE: Cell debris and dead cells were excluded from the analysis based on DRAQ5™ and DAPI fluorescence.
Figure 2. EasySep™-Isolated CD45+ Cells are Representative of the Starting Leukocyte Population
Mass cytometry data comparing the composition of immune subsets in PBMCs and EasySep™-isolated cells from the same donor. Starting with whole blood, PBMCs were prepared by density gradient centrifugation using Lymphoprep™. To compare immune subset composition pre- and post-EasySep™ isolation, a fraction of the PBMCs was further isolated using EasySep™ Release Human CD45 Positive Selection Kit and the pre- and post-isolated fractions were assessed by mass cytometry (CyTOF®). t-SNE plots of cells stained with 19 markers and analyzed by CyTOF® are shown (n = 1).
Figure 3. EasySep™-Isolated CD45+ Cells Produce IFN-gamma in Response to Antigen and Mitogen Stimulation
PBMCs pre- and post-EasySep™ isolation were incubated for 24 hours in the presence of peptide pools (CEF for antigen-specific CD8+ T cell response and CPI for antigen-specific CD4+ T cell response) or mitogen (phytohemagglutinin [PHA]). Following incubation, ELISpot plates were processed and IFN-gamma-producing cells were counted using an AID ELISpot reader. Representative images of ELISpot assays are shown (n = 3).
Figure 4. CD45+ Cells Isolated by EasySep™ from Various Tissues Are Highly Purified
A humanized mouse tumor model was generated by engraftment of human CD34+ hematopoietic stem and progenitor cells into NRG-3GS mice followed by xenotransplantation with human cancer cell lines, MDA-MB-231 (breast cancer) or SKOV-3 (ovarian carcinoma). Starting with a single-cell suspension of spleens, lungs, bone marrow, or tumors, human CD45+ leukocytes were isolated using EasySep™ Release Human CD45 Positive Selection Kit. The starting frequencies and isolated purities of human CD45+ cells of individual experiments and averages are shown.
Tebentafusp, a T cell engager, promotes macrophage reprogramming and in combination with IL-2 overcomes macrophage immunosuppression in cancer
Nature Communications 2025 Mar
Abstract
Uveal melanoma (UM) is the most common intraocular cancer in adults, with metastatic disease (mUM) occurring in approximately half of the patients. Tebentafusp, an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC), is a therapeutic shown to improve overall survival (OS) in HLA-A*02:01+ adult patients with mUM. Here we investigate the impact of tumor-associated macrophages (TAM) on ImmTAC activity. In vitro, M2 macrophages inhibit ImmTAC-mediated tumor-killing in a dose-dependent and contact-dependent manner. Accordingly, high baseline intratumoral TAM-to-T cell ratios correlate with shorter OS (HR = 2.09, 95% CI, 1.31–3.33, p = 0.002) in tebentafusp-treated mUM patients from a phase 2 trial. By contrast, IL-2 conditioning of T cells overcomes M2 macrophage-mediated suppression in vitro, while ImmTAC treatment leads to M2-to-M1 macrophage reprogramming both in vitro and in tebentafusp-treated mUM patients. Overall, we show that tebentafusp reshapes the tumor microenvironment to enhance anti-tumor T cell activity, whilst combining tebentafusp with IL-2 may enhance benefit in patients with high levels of TAM. ‘T cell engagers promote antitumor immunity, but how macrophage modulates this activity in tumor is still unclear. Here the authors show, using biopsies from patients with uveal melanoma and single cell analyses, that a T cell engager, tebentafusp, reprograms tumor-associated macrophages and ameliorates, in synergy with IL-2, immunosuppression to cancer.
A heterozygous CEBPA mutation disrupting the bZIP domain in a RUNX1 and SRSF2 mutational background causes MDS disease progression
Nature Communications 2025 Jul
Abstract
Myelodysplastic syndrome disease (MDS) is caused by the successive acquisition of mutations and thus displays a variable risk for progression to AML. Mutations in CEBPA are commonly associated with a high risk of disease progression, but whether they are causative for AML development is unclear. To analyse the molecular basis of disease progression we generated MDS patient-derived induced pluripotent stem cells from a low risk male patient harbouring RUNX1/SRSF2 mutations. This experimental model faithfully recapitulates the patient disease phenotypes upon hematopoietic differentiation. Introduction of a frameshift mutation affecting the C/EBPα bZIP domain in cells from low-risk stages mimicks disease progression by reducing clonogenicity of myeloid cells, blocking granulopoiesis and increasing erythroid progenitor self-renewal capacity. The acquisition of this mutation reshapes the chromatin landscape at distal cis-regulatory regions and promotes changes in cellular composition as observed by single cell RNAseq. Mutant C/EBPα is therefore causative for MDS disease progression. Our work identifies mutant CEBPA as causative for MDS disease progression, providing a new isogenic MDS experimental model for drug screening to improve diagnostic and therapeutic strategies. In Myeloiddysplastic syndromes, CEBPA mutations are linked to disease progression and AML. Here, the authors use somatic reprogramming and genome editing to generate isogenic cell lines from an MDS patient, identifying CEBPA bZIP domain disruption as causative for disease progression.
Influence of donor–recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation
HemaSphere 2024 May
Abstract
AbstractImmunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years, reproducibility, as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here, we used various immunodeficient mouse models to characterize the effect of donor–recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex‐specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly, human female donor cells display heightened sensitivity to the recipient mice's gender, influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics, shedding light on important considerations for future studies, particularly in the context of pre‐clinical research.
Mouse monoclonal IgG1 antibody against human, chimpanzee CD45
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EasySep™ Release Human CD45 Positive Selection Kit
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