We know that cell isolation is only part of your workflow. We designed this kit to isolate NK cells in as little as 8 minutes, so you can get to your downstream experiments as soon as possible.
Figure 1. Typical EasySep™ Human NK Cell Isolation Profile
Starting with human PBMCs, the NK cell (CD3-CD56+) content of the isolated fraction is typically 85.0 ± 8.0% (mean ± SD). In the above example, the final purities of the start and isolated fractions are 5.9% and 86.7%, respectively.
Figure 2. CD56+CD3− NK Cells Expand Over 14 Days in Feeder- and Serum-Free Culture Conditions
Human CD56+CD3− NK cells isolated using EasySep™ Human NK Cell Isolation Kit (Catalog #17955) were cultured using ImmunoCult™ NK Cell Expansion Kit (Catalog #100-0711) for 14 days. Cells were harvested and analyzed for expression of characteristic NK cell markers, including CD56, CD3, CD16, CD94, KIR, NKG2D, NKp46, NKp30, and NKp44 by flow cytometry. Staining for killer cell immunoglobulin-like receptor (KIR) molecules was performed using two different antibody clones, HP-MA4 and 180704, which recognize distinct KIR molecules. Dead cells were excluded by light-scatter profile and DRAQ7™ staining. (A - H) Representative flow cytometry plots. (I) The average frequencies of viable CD56+CD3− and CD56+CD16+ NK cells on Day 14 were 87 ± 1% and 75 ± 2%, respectively. The average fold expansion of CD56+CD3− cells was 89 ± 17. Results shown represent mean ± SEM (n = 34).
Figure 3. Expanded NK Cells Are Functional, Killing K562 Cells in Co-Culture
CD56+CD3− NK cells isolated using EasySep™ Human NK Cell Isolation Kit (Catalog #17955) were expanded using ImmunoCult™ NK Cell Expansion Kit (Catalog #100-0711) and co-cultured with Incucyte® Cytolight Rapid Dye-labeled K562 cells at a 1:1 ratio of NK:K562 cells at 37°C for 4 hours. Incucyte® Caspase-3/7 Dye, a caspase-inducible dye, was added to the co-culture to detect caspase-induced apoptosis of the K562 cells. Images were obtained every hour using the Incucyte® imaging system and then analyzed to determine % killing (# apoptotic K562 cells ÷ # total labeled K562 cells). After 4 hours, an average of 48 ± 2.4% K562 cells were killed (n = 9). Data represent mean ± SEM.
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.
Activation of human ?? T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells. M. Mata Forsberg et al. Journal of leukocyte biology 2022 mar
Abstract
Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, ?? T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-$\gamma$ expression in MAIT-, ?? T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate ?? T- and NK cells was fully dependent on the presence of both monocytes and $\alpha$$\beta$ T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.
The Combination of Radiotherapy and Complement C3a Inhibition Potentiates Natural Killer cell Functions Against Pancreatic Cancer. Q. H. Sodji et al. Cancer research communications 2022 jul
Abstract
Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.
C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models. S. Nandagopal et al. Cancer immunology research 2022 feb
Abstract
Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high-affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represent a new strategy to improve the efficiency of immunotherapy for cancer treatment.
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