Easily and efficiently isolate highly purified human CD8+ T cells from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or washed leukapheresis samples by immunomagnetic negative selection, with the EasySep™ Human CD8+ 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: CD4, CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, GlyA, and TCRgd. The magnetically labeled cells are then separated from the untouched desired CD8+ 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 8 minutes, the desired CD8+ T cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction.
This product replaces EasySep™ Human CD8+ T Cell Enrichment Kit (Catalog #19053) for even faster cell isolations.
For large-scale isolation of human CD8+ T cells from leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-0710).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Alternatively, choose ready-to-use, ethically sourced, primary Human Peripheral Blood CD8+ T Cells, Frozen isolated with EasySep™ Human CD8+ T Cell Isolation Kit. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
Figure 1. EasySep™ Human CD8+ T Cell Isolation Kit
Starting with human peripheral blood mononuclear cells (PBMCs), the CD8+ T cell content (CD3+CD8+) of the isolated fraction is typically 85.6 ± 4.9% (mean ± SD for the purple EasySep™ Magnet).
Figure 2. Experimental Workflow: DC/T Cell Co-Culture Protocol for the Activation and Expansion of Antigen-Induced CD8+ T Cells
The EasySep™ Human CD8+ T Cell Isolation Kit (Catalog #17953) can be used as a part of a workflow to assess antigen-specific T cell functionality by co-culturing dendritic cells (DCs) and CD8+ T cells. (1) Isolate monocytes from Human Peripheral Blood Leukopak, Fresh (Catalog #70500) or from Human Peripheral Blood Mononuclear Cells (PBMCs), Fresh or Frozen (Catalog #70025), using EasySep™ Human Monocyte Isolation Kit (Catalog #19359). (2) Culture monocytes to generate monocyte-derived dendritic cells (Mo-DCs) using ImmunoCult™ Dendritic Cell Culture Kit (Catalog #10985) and the peptide(s) of interest. (3) Isolate CD8+ T cells from the same donor’s blood or PBMCs using EasySep™ Human CD8+ T Cell Isolation Kit. (4) Co-culture DCs and CD8+ T cells in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981). (5) For short-term co-culture, isolate CD8+ T cells using EasySep™ Human CD8+ Cell Isolation Kit and label the isolated CD8+ T cells with a cell proliferation tracking dye. Set up co-culture by seeding peptide-pulsed dendritic cell suspension with the CD8+ T cell suspension at a 1:4 ratio. Harvest the co-cultures and quantify the antigen-specific CD8+ T cells with tetramer staining after 6 days. (6) For long-term co-culture, expand antigen-specific CD8+ T cells with additional supplements. Analyze the phenotype and function of expanded CD8+ T cells by assessing surface markers or cytokine production. Alternatively, enrich antigen-specific CD8+ T cells with EasySep™, rest the cells for 2 days, and then assess killing activity.
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™ be used for either positive or negative selection?
Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).
How does the separation work?
Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.
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™ separations be automated?
Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.
Can EasySep™ be used to isolate rare cells?
Yes. We recommend a cell concentration of 2x108 cells/mL and a minimum working volume of 100 µL. Samples containing 2x107 cells or fewer should be suspended in 100 µL of buffer.
Are the EasySep™ magnetic particles FACS-compatible?
Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.
Can the EasySep™ magnetic particles be removed after enrichment?
No, but due to the small size of these particles, they will not interfere with downstream applications.
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.
For positive selection, can I perform more than 3 separations to increase purity?
Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.
How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?
Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.
If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.
Discovery of potent immune-modulating molecule taccaoside A against cancers from structures-active relationships of natural steroidal saponins. Z. Dai et al. Phytomedicine : international journal of phytotherapy and phytopharmacology 2022 sep
Abstract
BACKGROUND In recent years, the T-cell therapy and immune checkpoint inhibitors toward CTLA-4 and PD-1/PD-L1 axis antibody therapy have acquired encouraging success. However, most of patients were still not benefited with lots of troubles, such as low penetration of tissues/cells, strong immunogenicity and cytokine release syndrome, and long manufacturing process and expensive costs. By contrast, the immune-modulating small molecules possessed natural advantages to overcome these obstacles and might achieve greater success. PURPOSE Exploring the potent immune-modulating natural small molecules and revealing what kinds of molecules or structures with the immunomodulatory activity against cancers. METHODS A novel non-cytotoxic T-cell immunomodulating screening model was used to identify the cytotoxic/selective/immunomodulatory bioactivity for 148 natural steroidal saponins. The structure-activity relationships (SARs) research was used to reveal the key groups for immunomodulation/cytotoxicity/selectivity. The negative selection was used to isolate and purify the T-cell. The cell viability assay was used to measure the anti-cancer effect in vitro. The ELISA assay was used to detect the cytokines for IL-1$\beta$, IL-6, TNF-$\alpha$, IFN-$\gamma$, IL-12, perforin and granzyme B (GZMB). The western blotting assay was used to research the immunomodulatory mechanism. The siRNA knockdown was used to generate the IFN-$\gamma$ resistant melanoma cells. The NOG immune-deficient mice were used to evaluate the anti-tumor efficacy in vivo. The peripheral blood samples from 10 cancer patients were used to detect the broad population anti-tumor efficacy. RESULTS It was reported that the correlation among structures and immunomodulation/ cytotoxicity/selectivity, in which opening ring-F with 26-O-glucopyranosyl, disaccharide and trisaccharide chains at C-3, steric hindrance and polarity of C-22 were key immunomodulatory groups. Moreover, taccaoside A was identified as the most potent candidate against cancer cells, including non-small cell lung cancer, triple negative breast cancer, and the IFN-$\gamma$ resistant melanoma, partly through enhancing T lymphocyte mTORC1-Blimp-1 signal to secrete GZMB. Besides, 10 patients derived T-cell also would be modulated against cancer cells in vitro. Moreover, the overall survival was great extended (>140 days vs 93 days) with nearly 100% tumor burden disappearance (0 mm3vs 1006 ± 79.5 mm3) in mice. CONCLUSION This work demonstrated one possibility for this concerned purpose, and identified a potent immune-modulating natural molecule taccaoside A, which might contribute to cancer immunotherapy in future.
CircRNA VIM silence synergizes with sevoflurane to inhibit immune escape and multiple oncogenic activities of esophageal cancer by simultaneously regulating miR-124/PD-L1 axis. C. Gao et al. Cell biology and toxicology 2022 oct
Abstract
BACKGROUND Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia, but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. METHODS Bioinformatic tools, luciferase assay, and RNA immunoprecipitation were used to examine regulations between circ-VIM, miR-124-3p (miR-124), and PD-L1. CCK-8, wound healing, and Transwell assays were used to measure cell proliferation, migration, and invasion, respectively. The impacts of EC cells on cytotoxicity, proliferation, and apoptosis of CD8+ T cells were examined using LDH assay, CFSE staining, and Annexin V/PI staining, respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. RESULTS Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane, independent of circ-VIM, also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis, silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro, and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. CONCLUSIONS Silencing circ-VIM and applying sevoflurane, by separately regulating miR-124/PD-L1 axis, presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells.
Role of CD8+ T cell exhaustion in the progression and prognosis of acute respiratory distress syndrome induced by sepsis: a prospective observational study. L. Yan et al. BMC emergency medicine 2022 nov
Abstract
BACKGROUND CD8+ T cells are important for protective immunity against intracellular pathogens. Excessive amounts of antigen and/or inflammatory signals often lead to the gradual deterioration of CD8+ T cell function, a state called exhaustion". However the association between CD8+ T cell exhaustion and acute respiratory distress syndrome (ARDS) has not been studied. This study was conducted to elucidate how CD8+ T cells and inhibitory receptors were related to the clinical prognosis of ARDS. METHODS A prospective observational study in an emergency department enrolled patients who were diagnosed with sepsis-associated ARDS according to the sepsis-3 criteria and Berlin definition. Peripheral blood samples were collected within 24??h post recruitment. CD8+ T cell count proliferation ratio cytokine secretion and the expression of coinhibitory receptors were assayed. RESULTS Sixty-two patients with ARDS met the inclusion criteria. CD8+ T cell counts and proliferation rates were dramatically decreased in non-surviving ARDS patients. Increasing programmed cell death 1 (PD-1) expression on the CD8+ T cell surface was seen in patients with worse organ function while an increasing level of T cell immunoglobulin mucin-3 (Tim-3) was associated with a longer duration of the shock. Kaplan-Meier analysis showed that low CD8+ T cell percentages and increased inhibitory molecule expression were significantly associated with a worse survival rate. CONCLUSIONS CD8+ T cells and coinhibitory receptors are promising independent prognostic markers of sepsis-induced ARDS and increased CD8+ T cell exhaustion is significantly correlated with poor prognosis."
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