Mouse nk cell isolation kit 2

Manufactured by Miltenyi Biotec

Sourced in Germany

The Mouse NK Cell Isolation Kit II is a magnetic cell separation product designed to isolate natural killer (NK) cells from mouse splenocytes. The kit utilizes a negative selection approach to enrich for NK cells by depleting non-NK cell populations.

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12 protocols using mouse nk cell isolation kit 2

TRAIL-deficient NK Cell Transfer in Mice

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Liver leukocytes were obtained from wild type B6 mice. Liver NK cells were then negatively separated by using a mouse NK cell isolation kit II (Miltenyi Biotec, Auburn, CA, USA). TRAIL⁻ NK cells were further sorted magnetically using biotin-conjugated anti-mouse CD253 (TRAIL; N2B2; eBioscience) and streptavidin microbeads (Miltenyi Biotec) in the negative fraction. The purity of isolated TRAIL⁻ NK cells was assessed by flow cytometry. The liver TRAIL⁻ NK cells were intravenously injected into Rag-2^−/− γ chain^−/− mice (0.5×10⁶ cells/mouse). The transferred mice were then divided into two groups. The fasted mice received only water and fed mice received both food and water for 3 days. The lymphocytes from the liver, spleen, and bone marrow of transferred or non-transferred (control) mice were harvested after the fasting period, and NK cell phenotyping was performed.

Dang V.T., Tanabe K., Tanaka Y., Tokumoto N., Misumi T., Saeki Y., Fujikuni N, & Ohdan H. (2014). Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation. PLoS ONE, 9(10), e110748.

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Activated NK Cell Adoptive Transfer

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Splenocytes from C57BL/6 (CD45.1⁺) mice were used to isolate donor NK cells. NK cells were enriched by using the mouse NK Cell Isolation Kit II (Miltenyi Biotec) and activated in vitro for 5 days with 5.000 U/mL IL-2 (Chiron) before intravenous (IV) infusion into C57BL/6 (CD45.2⁺) recipient mice.

Devillier R., Calmels B., Guia S., Taha M., Fauriat C., Mfarrej B., Venton G., Vivier E., Olive D., Chabannon C., Blaise D, & Ugolini S. (2021). Phase I Trial of Prophylactic Donor-Derived IL-2-Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor. Cancers, 13(11), 2673.

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Cytotoxicity Assay of NK Cells

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Natural killer cells were isolated from C57BL/6N mice spleens using the mouse NK cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. The cytotoxicity of NK cells was determined in a calcein release assay against cancer cells. The target cells, cancer cells, were labelled with calcein‐AM (#C396; Dojindo, Kumamoto, Japan) at 4 μM per 5 × 10⁵–10⁶ cells in 1 mL PBS for 25 min at 37°C. Labeled cancer cells were incubated with NK cells in a 96‐well plate with 200 μL culture medium for 4 h at 37°C and 5% CO₂. After centrifugation, 100 μL supernatant was collected, and the fluorescence was measured with excitation and emission wavelengths at 495/515 nm. The percentage of specific lysis was calculated as follows: 100× (experimental release − spontaneous release)/(maximum release − spontaneous release).

Li S., Nishikawa T, & Kaneda Y. (2017). Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule‐1 and enhances natural killer cell sensitivity on cancer cells. Cancer Science, 108(12), 2333-2341.

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ADCC Assay Protocol for NK and CD8+ T Cells

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ADCC assays were performed at an effector-to-target (E:T) ratio of 100:1, 10:1, or 1:1. The effector cells, NK cells and CD8a+ T cells, were isolated from single-cell suspensions of mouse spleen using the mouse NK Cell Isolation Kit II (Miltenyi) and the mouse CD8a+ T Cell Isolation Kit (Miltenyi). At first, the effector cells were stimulated with rmIL-18–stimulated B cells, IL-18BP–stimulated B cells, a combination of IL-18–stimulated B cells and PPI, or PPI only. Then the effector cells were co-cultured with target cells, 10,000 cells/well, in a U bottom 96-well plate. Twenty hours later, the supernatant was collected for lactate dehydrogenase (LDH) release measurements using LDH Cytotoxicity Assay Kit (Cayman, United States).

Zhao Y., Shen M., Feng Y., He R., Xu X., Xie Y., Shi X., Zhou M., Pan S., Wang M., Guo X, & Qin R. (2017). Regulatory B cells induced by pancreatic cancer cell-derived interleukin-18 promote immune tolerance via the PD-1/PD-L1 pathway. Oncotarget, 9(19), 14803-14814.

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Isolation of Mouse NK Cells

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Splenocytes were prepared by passing the spleen through a 100-µm nylon mesh and subsequent washing with ice-cold phosphate-buffered saline (PBS). Erythrocytes were removed from splenocytes by gradient centrifugation with Ficoll-Paque Plus (GE Healthcare Europe, Munich, Germany). NK cells were enriched from purified splenocytes using the mouse NK cell isolation kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The purity of isolated CD3⁻NK1.1⁺ NK cells was monitored by flow cytometry and varied between 60 and 85%.

Koch C., Kim Y., Zöller T., Born C, & Steinle A. (2017). Chronic NKG2D Engagement In Vivo Differentially Impacts NK Cell Responsiveness by Activating NK Receptors. Frontiers in Immunology, 8, 1466.

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Cell Culture and Isolation Protocols

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The cell lines NS-1 (mouse myeloma) and 300.19 (mouse pre-B) were obtained from the American Type Culture Collection. Cells were grown in RPMI 1640 medium (GIBCO-BRL, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO), 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine (GIBCO-BRL). 300.19 cells were maintained in media supplemented with 0.05 mM 2-mercaptoethanol (GIBCO-BRL). Primary mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL) supplemented as indicated above. Primary macrophages were elicited from peritoneal exudate cells (PECs) following i.p. injection of 1 ml of 3% thioglycollate (Sigma Aldrich) into BALB/c mice. PECs were removed by peritoneal lavage. Cells were plated out at 2×10⁵ cells/ml in supplemented RPMI 1640 medium, and incubated for 2 h at 37°C, 5% CO₂, after which nonadherent cells were washed away with phosphate buffered saline (PBS). Macrophage preparations were confirmed by flow cytometry using the markers F4/80 and CD11b (about 95% were F4/80⁺CD11b⁺). NK cells were obtained from mouse spleen using the mouse NK cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) on an AutoMACS (Miltenyi Biotec).

Zarama A., Pérez-Carmona N., Farré D., Tomic A., Borst E.M., Messerle M., Jonjic S., Engel P, & Angulo A. (2014). Cytomegalovirus m154 Hinders CD48 Cell-Surface Expression and Promotes Viral Escape from Host Natural Killer Cell Control. PLoS Pathogens, 10(3), e1004000.

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Purification and Culture of Murine NK Cells

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NK cells were purified under sterile conditions from spleens at gestational day 10.5 using a mouse NK cell isolation kit II (130-096-892; Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The purity of the NK cells routinely exceeded 95%, as determined using flow cytometry.^{12 (link), 13 (link)}NK cells were seeded in suspension multiple-well plates and cultured in RPMI 1640 complete medium supplemented with 10% FBS (10099-141; Gibco, Australia), 100 U/ml penicillin, 100 µg/mL streptomycin (SV30010; HyClon, Logan, UT, USA) and 200 U/ml recombinant murine IL-2 (212-12; Peprotech, Rocky Hill, NJ, USA). Cells were seeded at a concentration of 1×10⁶ cells/ml and grown at 37°C in a humidified incubator with 5% CO₂.
GC7 stock solution was diluted with complete medium to obtain the indicated concentrations before use. GC7 is a substrate of serum amine oxidase (SAO); therefore, to ensure SAO to be inhibited, aminoguanidine (396494; Sigma-Aldrich, St Louis, MO, USA), an inhibitor of SAO, was added at a concentration of 1 mM to all cultures (including in the control group).^{14 (link)} In subsequent experiments, the final GC7 concentrations were 20, 30 and 40 µM, respectively. An equal volume of solvent was used as a negative control.

Qin X., Liu X., Shan B., Shi L., Sharma S., Wu J, & Lin Y. (2014). Inhibition of eIF5A results in aberrant uterine NK cell function and embryo loss in mice. American journal of reproductive immunology (New York, N.Y. : 1989), 71(3), 229-240.

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NK Cell Activation and Cytokine Production

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Single-cell suspensions of spleen from naive WT, GrzM^−/−, pfp^−/− or GrzB^−/−mice were enriched for NK cells (NK1.1⁺/CD3⁻) by negative selection with a mouse NK cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted to purity (BDAriaII, San Jose, CA, USA). Human NK cells were enriched from Buffy-coats (EasySep negative enrichment kit, Stemcell Technologies, Vancouver, BC, Canada; 19055). Mouse or human NK cells were stimulated with 1 ng IL-12/ml and 10 ng IL-15/ml, 1 ng IL-12/ml and 5 ng IL-18/ml, or 1000 U/ml IL-2 and 1 ng IL-12/ml for 3, 6 and 24 h or for 4 and 24 h. Cells were used for surface marker staining, intracellular staining or to extract RNA for RT-PCR; supernatants were harvested for CBA assays or were analysed by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Baschuk N., Wang N., Watt S.V., Halse H., House C., Bird P.I., Strugnell R., Trapani J.A., Smyth M.J, & Andrews D.M. (2014). NK cell intrinsic regulation of MIP-1α by granzyme M. Cell Death & Disease, 5(3), e1115-.

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Isolation and Characterization of Murine NK Cells

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Mouse splenocytes obtained from naïve C57BL/6 mouse spleens were made in a single cell suspension through mechanical methods and strained through a 35 µm mesh. Then, the mouse NK Cell Isolation Kit II (MACS-Miltenyi Biotec) was used following the manufacturer’s protocol. Purified murine NK cells were collected in RPMI (10% FBS, 50 mM βME, 5 IU/ml of mIL-2 (Biolegend), and 2 ng/ml of mIL-15 (Biolegend) and used immediately for ADCC at either 1:5, 1:10, or 1:20 target to effector cell ratio (see below). Remaining NK cells were stained with 1:200 dilutions of anti-CD3, NK1.1, CD335 (NKp46), CD32/16 markers (BioLegend, catalogue number: 100221; 108709; 137611; 101323), and by 1:1000 dilution of Zombie® UV viability dye (BioLegend) and characterized by flow cytometry (BD LSRFortessa) to confirm NK cell purity and Fcγ-Receptor III expression^{29 (link)}.

Abreu-Mota T., Hagen K.R., Cooper K., Jahrling P.B., Tan G., Wirblich C., Johnson R.F, & Schnell M.J. (2018). Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever. Nature Communications, 9, 4223.

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Isolation and Purification of Mouse Liver Mononuclear Cells

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LMNCs were prepared as previously described^{50 (link)}. Briefly, the liver was removed after perfusion with 1 mL phosphate-buffered saline (PBS) supplemented with 10% heparin via the portal vein. LMNCs were obtained from the liver by perfusion with 50 mL PBS supplemented with 0.1% ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO, USA). For in vitro culturing, LMNCs were separated into NK and non-NK cell fractions using the Mouse NK Cell Isolation Kit II (Miltenyi Biotec). Only the samples with more than 90% purity of isolated fractions were used for further analyses.

Saeki Y., Ishiyama K., Ishida N., Tanaka Y, & Ohdan H. (2019). Memory-like Liver Natural Killer Cells are Responsible for Islet Destruction in Secondary Islet Transplantation. Scientific Reports, 9, 1022.

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