Easysep mouse nk cell isolation kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep Mouse NK Cell Isolation Kit is a laboratory product designed to isolate natural killer (NK) cells from mouse splenocytes or bone marrow. The kit utilizes a positive selection method to enrich the target cell population.

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EasySep cell isolation kit (6 citations) NK isolation kit (4 citations) EasySep isolation kits (5 citations) Cell isolation kits (4 citations) EasySep kits (21 citations) Isolation kits (7 citations)

53 protocols using easysep mouse nk cell isolation kit

Isolation and Activation of Murine NK Cells

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NK cells were isolated from the spleens of C57BL/6 mice via negative magnetic selection using the EasySep™ mouse NK Cell Isolation Kits and EasySep™ Magnet (STEMCELL Technologies Inc., Vancouver, BC, Canada). CD3⁻CD49b⁺ NK cell purity was 94.1% after the NK cell isolation from the spleen cells, but CD3⁺ T cells were reduced from 27.3% to 0.28% after the isolation (Supplementary Figure S2). To generate the activated NK (ANK) cells, purified NK cells (5 × 10⁵ cells/mL) were cultured in 6-well plates for 48 h in 10% FBS, 1× antibiotics, 1× sodium pyruvate, 1× non-essential amino acids, and 1 × 2-mercaptoethanol containing RPMI 1640 media with GlutaMax (10% LCM) in the presence of 100 ng/mL MPL, 1000 ng/mL poly I:C, the combination of 100 ng/mL MPL and 1000 ng/mL poly I:C, and only the medium. The ANK cells were then harvested, washed to remove all excess reagents, and resuspended in 10% LCM before use in future experiments.

Le C.T., Ahn S.Y., Kang S.M, & Ko E.J. (2021). Functional NK Cell Activation by Ovalbumin Immunization with a Monophosphoryl Lipid A and Poly I:C Combination Adjuvant Promoted Dendritic Cell Maturation. Vaccines, 9(10), 1061.

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Evaluating NK Cell-Mediated Cytotoxicity

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NK cells were isolated from splenocytes of C57Bl/6J mice with the EasySep Mouse NK Cell Isolation Kits (Stemcell Technologies). Population purity assessed by flow cytometry was >95%. B16-GFP cells were allowed to adhere overnight before purified splenic NK cells from C57Bl/6J mice were added at an Effector:Target ratio of 20:1. Doses of FIST15 or control cytokines (0.32pM-1000pM) were added to the co-culture for 48 hours. Floating cells and debris were washed away and adherent cells were trypsinized, washed, and analyzed for GFP positivity. %B16-GFP⁺ survival was calculated by dividing the number of GFP⁺ events in each condition by GFP⁺ events from B16-GFP cultures without NK cells. MC-38 cytotoxicity assay was conducted in a similar manner. MC-38 cells were pre-labeled with CFSE and %MC-38 survival was calculated on the basis of CFSE⁺ events. Event counts were normalized to AccuCheck counting beads (Thermo Fisher). Granzyme B and caspase 6 serine protease activity was assayed by PanToxiLux cytotoxicity assay kit (OncoImmunin). Co-cultures of NK cells and B16-F0 with FIST15 or controls were set up as described above. 24 hours post co-culture, fluorogenic substrate was added to the culture for 2 hours, after which cells were trypsinized, washed, and stained with CD45. Substrate cleavage was assayed by flow cytometry to determine (FL-1 fluorescence) on CD45^×, B16-F0 cells.

Ng S., Deng J., Chinnadurai R., Yuan S., Pennati A, & Galipeau J. (2016). Stimulation of natural killer cell-mediated tumor immunity by an IL-15/TGF-ß neutralizing fusion protein. Cancer research, 76(19), 5683-5695.

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Enhanced NK Cell Cytotoxicity Against Tumors

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Example 1

NK cells are isolated from the mice using the EasySep™ Mouse NK Cell Isolation Kit (STEMCELL™ Technologies) following the manufacturer's protocol. Isolated NK cells are incubated with the culture medium containing IFN-alpha (Lee Biomolecular, San Diego, Calif.; 1×10³U/1×10⁶cells) at 37° C. and 5% CO₂for 7-30 days. The culture medium is changed every 3 days with fresh IFN-alpha. Every 5 days, expanded NK cells are transferred to T25 flask or T75 flasks at a concentration of 0.5×10⁶cells.

Nude mice are injected with HLA-negative tumor cells (subcutaneous injection) to form a tumor that expresses no or reduced levels of HLA.

One to three days after the formation of tumors in the mice, the mice are injected via intravenous injection with 5×10⁶NK cells that are treated with IFN-alpha. The mice injected with the untreated NK cells are used as control. Tumor growth in mice injected with treated NK cells is delayed compared to mice injected with untreated NK cells.

US11571469B2. Methods of treating cancer with interferon wherein the cancer cells are HLA negative or have reduced HLA expression (2023-02-07). Mayo Foundation for Medical Education and Research [US]. Inventors: Svetomir N. Markovic [US].

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Epa1, Epa4, Epa6, and Epa7 Ectopic Expression

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All Cg strains used in the paper were derived from BG2 (Cormack and Falkow, 1999 (link)). For ectopic expression of
Epa1, Epa4, Epa6 and Epa7 Saccharomyces cerevisiae cells were
used as previously described (Zupancic et al.,
2008). Murine NK cells were prepared from BALB/c mice using the
EasySep Mouse NK cell Isolation kit (Stemcell). See also Supplemental Information.

Vitenshtein A., Charpak-Amikam Y., Yamin R., Bauman Y., Isaacson B., Stein N., Berhani O., Dassa L., Gamliel M., Gur C., Glasner A., Gomez C., Ben-Ami R., Osherov N., Cormack B.P, & Mandelboim O. (2016). NK cell recognition of Candida glabrata through binding of NKp46 and NCR1 to fungal ligands Epa1, Epa6, and Epa7. Cell host & microbe, 20(4), 527-534.

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Isolation and Culture of Tumor-Associated NK Cells

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Primary splenocytes were isolated by mechanical dissociation from spleens of WT FVB/n, MMTV-PyMT, or C3(1)-Tag mice or from the tumors of MMTV-PyMT mice. NK cells isolated from WT FVB/n mice were defined as hNK cells, and those isolated from tumor-bearing mice were defined as teNK cells. NK cells isolated from MMTV-PyMT tumors were defined as tiNK cells. The NK cells were purified from isolated splenocytes using EasySep Mouse NK Cell Isolation Kit per the manufacturer’s protocol (19855; StemCell Technologies). The isolated cells were then positively selected by FACS by gating on CD49b⁺/CD3⁻ cells and CD45⁺/CD49b⁺/CD3⁻ for NK cells isolated from tumors. NK cells were cultured in medium at 37°C containing 200 U/ml recombinant mouse IL-2 (402-ML-100; R&D Systems), 5 ng/ml recombinant mouse IL-15 (447-ML-010; R&D Systems), or 5 ng/ml recombinant mouse IL-15:IL-15R complex (14-8152-62; Thermo Fisher Scientific) for 16 h before use in assays. Pretreatment of teNK cells was performed with decitabine and azacitidine at 1-µM doses for 24 h (NCI Development Therapeutics Program).

Chan I.S., Knútsdóttir H., Ramakrishnan G., Padmanaban V., Warrier M., Ramirez J.C., Dunworth M., Zhang H., Jaffee E.M., Bader J.S, & Ewald A.J. (2020). Cancer cells educate natural killer cells to a metastasis-promoting cell state. The Journal of Cell Biology, 219(9), e202001134.

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Isolation of NK Cells from Poly I:C-Treated Mice

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Five C57Bl/6 mice were injected with 200 µg of poly I:C per injection and resuspended in sterile water (Invivogen #tlrl-picw) 1 day before isolating NK cells. Spleens from C57Bl/6 mice were collected and minced into fibrous tissue using a plunger. Splenocytes were collected with 5 mL of DMEM and passed through into a 100 µm cell strainer. 5 mL of ACK lysing buffer (Gibco #A1049201) was added and mixed for 5 min to lyse red blood cells. Then, mouse NK cells were isolated using EasySep Mouse NK cell isolation kit (Stemcell Technologies #19855), and flow cytometric analyses were performed to determine the percentage of NK1.1 positive NK cells.

Yun J., Saddawi-Konefka R., Goldenson B., Al-Msari R., Bernareggi D., Thangaraj J.L., Tang S., Patel S.H., Luna S.M., Gutkind J.S, & Kaufman D. (2024). CHMP2A regulates broad immune cell-mediated antitumor activity in an immunocompetent in vivo head and neck squamous cell carcinoma model. Journal for Immunotherapy of Cancer, 12(5), e007187.

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Murine NK Cell Activation Assay

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The mAbs APC anti-mouse CD49b (clone DX5) and Annexin V/7-AAD kit were obtained from BD PharMingen (San Diego, CA, USA). FITC anti-mouse CD3ε (clone 145-2C11), PE/CY7 anti-mouse CD19 (clone 1D3), and PE anti-mouse NK1.1 (clone PK136) were obtained from Sungene Biotech (Tianjin, China). PE/Cy7 anti-mouse CD69 (clone H1.2F3), PE/Cy7 anti-mouse NKp46 (clone 29A1.4), and FITC anti-mouse NKG2A/C/E (clone 20d5) were from eBioscience (San Diego, CA, USA). FITC anti-mouse granzyme B (clone GB11), PE anti-mouse NKG2D (clone CX5), PE anti-mouse perforin (clone S16009A), PE anti-mouse IFN-γ (clone XMG1.2), PE anti-mouse FasL (clone MFL3), PerCP/Cy5.5 anti-mouse TRAIL (clone N2B2), FITC anti-mouse Ly49A (clone YE1/48.10.6), and buffers for staining, fixation, and permeabilization, as well as CFSE Cell Division Tracker Kit, were from BioLegend (San Diego, CA, USA). Mouse IFN-γ ELISA kit was from Dakewe Biotech Co., Ltd. (Shenzhen, China). Recombinant murine IL-2, IL-12, and IL-15 were from PeproTech Inc. (Rockhill, NJ, USA). Poly(I:C) was from InvivoGen (San Diego, CA, USA). EasySep™ mouse NK cell isolation kit was from STEMCELL Technologies, Inc. (Cambridge, UK).

Miao M., Masengere H., Yu G, & Shan F. (2021). Reevaluation of NOD/SCID Mice as NK Cell-Deficient Models. BioMed Research International, 2021, 8851986.

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Expansion of Murine NK Cells

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Murine NK cells (either from C57BL/6 or Nu mice) were isolated from mouse splenocytes by immunomagnetic negative selection method via an EasySep mouse NK cell isolation kit (STEMCELL Technologies; catalog number, 19855) according to the manufacturer’s instructions. A poll of five healthy donors was used in each set of the in vitro study. Naïve NK cells were expanded by culturing in RPMI 1640 medium supplemented with 10% v/v of FBS, Anti-Anti (1×), 2 mM GlutaMAX supplement (Gibco), 0.1 mM nonessential amino acid (Gibco), 50 μM 2-mercaptoethanol (Sigma-Aldrich), and recombinant mouse IL-2 (500 U/ml) (R&D Systems; catalog number, 402-ML). The purities of CD3e⁻ and CD49b⁺ expanded NK cells isolated from C57BL/6 and Nu mice were above 95%, as quantified by FACS.

Au K.M., Park S.I, & Wang A.Z. (2020). Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy. Science Advances, 6(27), eaba8564.

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Murine NK Cell Isolation and Stimulation

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NK cells were purified from mouse splenocytes by using EasySep Mouse NK cell isolation kit (StemCell Techonologies, Vancouver, Canada) as described by the manufacturer. Cell purity was over 90% as confirmed by flow cytometry. Cells were plated and stimulated with IL-18 (R&D Systems, Minneapolis, MN) and/or IL-12 (PeproTech, Rocky Hill, NJ) for 6 hs before removing the supernatant for CBA analysis and staining the cells for ICS as described above.
For in vivo NK depletion, 100μg of α-NK1.1 antibody (Clone PK136, BioXCell, West Lebanon, NH) was intraperitoneally injected and NK cell depletion confirmed by analyzing blood samples 24hr later. Mice were primed with ΔactA-QVac, serum IFN-γ levels measured at 24hr post-infection and spleens removed and processed for ICS 7 days later as described.

Alice A.F., Kramer G., Bambina S., Baird J.R., Bahjat K.S., Gough M.J, & Crittenden M.R. (2017). Amplifying IFNγ signaling in DC by CD11c-specific loss of SOCS1 increases innate immunity to infection while decreasing adaptive immunity. Journal of immunology (Baltimore, Md. : 1950), 200(1), 177-185.

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Isolation of Mouse Splenic NK Cells

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Isolation of the mouse splenic NK cells was performed using the EasySep Mouse NK Cell Isolation Kit (Cat# 19855, StemCell Technologies, Vancouver, BC, Canada). Isolated NK cells were incubated in 1% FBS-RPMI-1640 at 37 °C in 5% CO₂.

Wang Y., Liu Z., Qi Y., Wu J., Liu B, & Cui X. (2024). Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling. Cells, 13(9), 728.

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