Before performing cell isolation using EasySep?, consult the product information sheet (PIS) to determine whether red blood cell (RBC) lysis is required for your sample type. RBC lysis should only be performed if indicated in the PIS. It is often recommended for blood samples; however, RBC lysis is not recommended for mouse splenocytes as it may reduce cell recovery. For the most accurate cell recovery calculation, we recommend performing total nucleated cell (TNC) count.
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Easily and efficiently isolate highly purified mouse T cells from single-cell suspensions of splenocytes or other tissues by immunomagnetic negative selection, with the EasySep? Mouse T Cell Isolation 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? negative selection procedure, unwanted cells are labeled with antibody complexes and magnetic particles. Unwanted cells expressing the following markers are targeted for removal: CD11b, CD45R, Ter119, CD49b, CD19, and CD24. The magnetically labeled cells are then separated from the untouched desired mouse T cells by using an EasySep? magnet and simply pouring or pipetting the desired cells into a new tube. Following magnetic cell isolation in as little as 15 minutes, the desired T cells are ready for downstream applications such as flow cytometry, culture, and cell-based experiments.
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.
Figure 1.Typical EasySep? Mouse T Cell Isolation Profile
Starting with mouse splenocytes, the T cell content (CD3+CD19-) of the isolated fraction is 96.6 ± 2.0% (mean ± SD), using the purple EasySep? magnet.
Figure 2.Cell Isolation Protocol Lengths
Typical time taken (in minutes) to isolate cells using select EasySep? kits.
Figure 3.ImmunoCult? Mouse T Cell Activator Kit Supports High Viability of Activated T Cells
Mouse T cells were isolated using EasySep? Mouse T Cell Isolation Kit (Catalog #19851), stimulated with ImmunoCult? Mouse T Cell Activator Kit (Catalog #100-1572), and cultured in IMDM + FBS formulation. Following 3 days of culture, the mean ± SD frequency of CD25+ cells was 91.9 ± 5.1% (n = 11) or 99.9 ± 0.1% (n = 5), when stimulated with ImmunoCult? Mouse CD3/CD28 T Cell Activator or ImmunoCult? Mouse CD3/CD28/CD2 T Cell Activator, respectively. Stimulated mouse T cells maintained expression levels of CD25 throughout the 7-day culture period.
Figure 4.Robust Expansion of EasySep?-Isolated Mouse T Cells Can Be Achieved Following Stimulation with ImmunoCult? Mouse T Cell Activator Kit
Mouse T cells isolated using EasySep? Mouse T Cell Isolation Kit (Catalog #19851) were expanded with ImmunoCult? Mouse T Cell Activator Kit (Catalog #100-1572) in IMDM + FBS formulation over 7 days. The number of viable cells was assessed every 2 - 3 days, and fresh medium supplemented with IL-2 was added. No additional ImmunoCult? Mouse T Cell Activator was added during the 7-day culture period. After 7 days in culture with ImmunoCult? Mouse CD3/CD28 T Cell Activator or ImmunoCult? Mouse CD3/CD28/CD2 T Cell Activator, stimulation resulted in a fold expansion of 23 ± 3.4 or 29.3 ± 4.8 (mean ± SEM, n = 6), respectively.
Figure 5.High Cell Proliferation is Observed in EasySep?-Isolated T cells After Stimulation with ImmunoCult? Mouse T Cell Activator
Mouse T cells isolated using EasySep? Mouse T Cell Isolation Kit (Catalog #19851) were labeled with CFDA-SE (Catalog #75003), stimulated with ImmunoCult? Mouse T Cell Activator Kit (Catalog #100-1572), and cultured in cultured in IMDM + FBS formulation. On Day 3, cells were harvested, stained with anti-mouse CD4 and CD8a antibodies, then measured by flow cytometry. Shown are CFDA-SE-labeled mouse T cells, gated on viable CD4+ (A) or CD8a+ (B) cells, cultured with no activator (top panel), with ImmunoCult? Mouse CD3/CD28 T Cell Activator (middle panel), or with ImmunoCult? Mouse CD3/CD28/CD2 T Cell Activator (bottom panel). Due to cell proliferation, the intensity of CFDA-SE signal is reduced by 50% for each cell division.
This product is designed for use in the following research area(s) as part
of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we
offer to support each research area.
Can EasySep™ Streptavidin RapidSpheres™ be used for either positive or negative selection?
Currently, EasySep™ Streptavidin RapidSphere™ kits are only available for negative selection and work by targeting and removing unwanted cells.
How does the separation work?
Streptavidin RapidSphere™ magnetic particles are crosslinked to unwanted cells using biotinylated antibodies. When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a new tube.
Which columns do I use?
The EasySep™ procedure is column-free. That's right - no columns!
How can I analyze the purity of my enriched sample?
The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.
Can EasySep™ Streptavidin RapidSphere™ separations be automated?
Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.
Are cells isolated using EasySep™ RapidSphere™ products FACS-compatible?
Yes. Desired cells are unlabeled and ready to use in downstream applications, such as FACS analysis.
Can I alter the separation time in the magnet?
Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.
Protocol for immunomagnetic enrichment of T cells from complex murine tissues
E. Trolio et al.
STAR Protocols 2026 Mar
Abstract
SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights?Isolation of leukocytes from murine BM, liver, heart and kidneys?Non-enzymatic dissociation of kidney and heart tissue?Protocol for immunomagnetic enrichment of T cells?Flow cytometry analysis of T cell purity and viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses.
Efficacy and safety of autologous CD5-KO anti-CD5 CAR-T cells in relapsed/refractory CD5+ hematological malignancies
J. Cheng et al.
Cell Reports Medicine 2026 Feb
Abstract
Chimeric antigen receptor (CAR)-T cell therapy targeting antigens shared with normal T cells requires genetic modifications to prevent fratricide. This phase 1 trial evaluates autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ hematologic malignancies. The overall response rate is 85.7%, including four complete responses. All patients experience cytokine release syndrome (six grade 1–2, one grade 3), and two patients develop immune effector cell-associated neurotoxicity syndrome. The most common grade ≥3 adverse events are cytopenia and infection, with unique observations of rash and autoimmune-related events. Post-infusion immunophenotyping shows persistent depletion of CD5+ T cells and CD19+ B cells, with reduced CD4/CD8 ratios. The human CD5 knockin murine model reveals skin lesions without significant vital organ involvement. These findings demonstrate CT125A’s therapeutic potential in CD5+ malignancies while highlighting the need for safety optimization. The trial has been registered at ClinicalTrials.gov (NCT04767308). Graphical abstract Highlights?CT125A achieves 85.7% response rate in relapsed/refractory CD5+ malignancies?CD5 gene deletion prevents fratricide and enhances CAR-T cell persistence?Prolonged CD5+ T cell aplasia associates with infections and autoimmune events?Mouse model reveals on-target, off-tumor effects primarily affecting skin tissue Cheng et al. report a phase 1 trial of autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ malignancies. CT125A achieves an 85.7% response rate but causes prolonged immunosuppression, infections, and autoimmune events, highlighting the need for safety optimization strategies.
Bi-specific T cell-engaging antibody triggers protective immune memory and glioma microenvironment remodeling in immune-competent preclinical models
M. Zannikou et al.
Journal for Immunotherapy of Cancer 2025 Oct
Abstract
Bispecific T cell-engagers (BTEs) are engineered antibodies that redirect T cells to target antigen-expressing tumors. BTEs targeting tumor-specific antigens such as interleukin 13 receptor alpha 2 (IL13RA2) and epidermal growth factor receptor variant III (EGFRvIII) have been developed for glioblastoma (GBM). However, there is limited mechanistic understanding of the action of BTE since prior studies were mostly conducted in immunocompromised animal models. To close this gap, the function of BTEs was assessed in the immunosuppressive tumor microenvironment (TME) of orthotopic and genetically engineered mouse models (GEMM) with intact immune systems. Method: sA BTE that bridges CD3 epsilon on murine T cells to IL13RA2-positive GBM cells was developed, and the therapeutic mechanism was investigated in immunocompetent mouse models of GBM. Multicolor flow cytometry, single-cell RNA sequencing (scRNA-seq), multiplex immunofluorescence, and multiparametric MRI across multiple preclinical models of GBM were used to evaluate the mechanism of action and response. Results: BTE-mediated interactions between murine T cells and GBM cells triggered T cell activation and antigen-dependent killing of GBM cells. BTE treatment significantly extended the survival of mice bearing IL13RA2-expressing orthotopic glioma and de novo forming GBM in the GEMM. Quantified parametric MRI validated the survival data, showing a reduction in glioma volume and decreased glioma viability. Flow cytometric and scRNA-seq analyses of the TME revealed robust increases in activated and memory T cells and decreases in immunosuppressive myeloid cells within the brains of mice following BTE treatment. Conclusions: Our data demonstrate that the survival benefits of BTEs in preclinical models of glioma are due to the ability to engage the host immune system in direct killing, induction of immunological memory, and modulation of the TME. These findings provide a deeper insight into the mechanism of BTE actions in GBM.
Hamster (Armenian) monoclonal IgG1 antibody against mouse CD3e
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EasySep? Mouse T Cell Isolation Kit
Before performing cell isolation using EasySep?, consult the product information sheet (PIS) to determine whether red blood cell (RBC) lysis is required for your sample type. RBC lysis should only be performed if indicated in the PIS. It is often recommended for blood samples; however, RBC lysis is not recommended for mouse splenocytes as it may reduce cell recovery. For the most accurate cell recovery calculation, we recommend performing total nucleated cell (TNC) count.
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