Efficiently deplete human CD45+ cells from fresh or previously frozen human peripheral blood mononuclear cell (PBMC) samples by immunomagnetic selection, with the EasySep™ Human CD45 Depletion Kit II. 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.
This straightforward, optimized EasySep™ procedure involves labeling cells with antibody complexes recognizing CD45 and magnetic particles. Labeled cells are separated from untouched cells using an EasySep™ magnet and by simply pouring off the unlabeled cells. The CD45+ cells remain in the tube. Following magnetic cell isolation, desired cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction. The CD45 antigen is expressed on all hematopoietic cells except erythrocytes and platelets.
This product replaces the EasySep™ Human CD45 Depletion Kit (Catalog #18259) for even faster cell isolations.
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 culture media, supplements, antibodies, and more.
Area of Interest Cancer, Immunology, Stem Cell Biology
Data Figures
Figure 1. Typical EasySep™ Human CD45 Depletion Profile
In the example above, CAMA cells were seeded into PBMCs at a starting frequency of 1.1% (98.9% CD45+; gated on DRAQ5™ for nucleated cells). The CAMA cell (EpCAM+) content of the depleted fraction is 98%, which is a 4.0 log depletion of CD45+ cells. NOTE: EpCAM is an antibody against an epithelial cell surface antigen expressed on CAMA cells.
Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia. A. Tuval et al. Haematologica 2022 nov
Abstract
Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC suggesting that while a pre-leukemic mutation can predispose for P53 misfolding additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53 specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention."
CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment. M. Seyfrid et al. Journal for immunotherapy of cancer 2022 jan
Abstract
PURPOSE Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.
Association of early changes of circulating cancer stem-like cells with survival among patients with metastatic breast cancer. P.-H. Chang et al. Therapeutic advances in medical oncology 2022
Abstract
BACKGROUND This study aimed to investigate the role of circulating tumor cells (CTCs) and circulating cancer stem-like cells (cCSCs) before and after one cycle of chemotherapy and assessed the effects of early changes in CTCs and cCSCs on the outcomes of patients with metastatic breast cancer. METHODS Patients with stage IV invasive ductal carcinoma of the breast who received first-line chemotherapy between April 2014 and January 2016 were enrolled. CTCs and cCSCs were measured before the first cycle of chemotherapy (baseline) and on day 21, before the second cycle of chemotherapy commenced; a negative selection strategy and flow cytometry protocol were employed. RESULTS CTC and cCSC counts declined in 68.8 and 45.5% of patients, respectively. Declines in CTCs and cCSCs following the first chemotherapy cycle were associated with superior chemotherapy responses, longer progression-free survival (PFS), and longer overall survival (OS). An early decline in cCSCs remained an independent prognostic indicator for OS and PFS in multivariate analysis. CONCLUSIONS A cCSC decline after one cycle of chemotherapy for metastatic breast cancer is predictive of a superior chemotherapy response and longer PFS and OS, implying that cCSC dynamic monitoring may be helpful in early prediction of treatment response and prognosis.
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