Nk macs isolation kit

Manufactured by Miltenyi Biotec

The NK MACS Isolation Kit is a laboratory equipment designed for the isolation and enrichment of natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs). The kit utilizes magnetic-activated cell sorting (MACS) technology to efficiently separate the desired NK cell population from the complex cellular mixture.

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Lab products found in correlation

Biocoll, by Merck Group (3 mentions) Recombinant human il 15, by Immunotools (3 mentions) T175 flask, by Sarstedt (2 mentions) Trisodium citrate blood collection tubes, by Sarstedt (2 mentions) Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions) Recombinant human il 2, by Immunotools (1 mentions) Human cd3 microbeads, by Miltenyi Biotec (1 mentions) Interleukin 2 (il 2), by Abcam (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions) Vi cell xr, by Beckman Coulter (1 mentions) Automacs separator, by Miltenyi Biotec (1 mentions) Lymphoprep, by STEMCELL (1 mentions) Pha m, by Merck Group (1 mentions)

Spelling variants of this product

MACS isolation kit (27 citations) NK isolation kit (25 citations) Isolation kits (10 citations) MACS kits (8 citations) NK MACS (6 citations)

6 protocols using nk macs isolation kit

Isolation of NK Cells from Donor Blood

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Healthy donor blood collected in trisodium citrate blood collection tubes was diluted with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco, Life Technologies Limited) in 1:1 ratio and overlayed on a 1.077g/mL separation medium (Biocoll, Merck Millipore). Density centrifugation was performed at room temperature (400g for 30min) without acceleration and brake. PBMCs were collected and washed with PBS followed by centrifugation at 300g for 8min at room temperature. Then plastic adherence was performed to deplete monocytes by incubating them in a T175 flask (Sarstedt) at 37°C and 5% CO2 for 1 hour. For NK isolation NK MACS Isolation Kit (Miltenyi Biotec) was used according to the manufacturer’s instructions. Purity of isolated NK cells was routinely tested and ranged from 90% to 97%. After isolation 5 U/ml recombinant human IL-15 (50µg, Immuno Tools) was added to NK cells that were used for functional experiments after overnight incubation.
Use of peripheral blood from healthy donors was approved by the institutional review board of the Medical Faculty of the University of Duisburg-Essen (approval number 07-3500 and 08-3590) and each donor signed an informed consent form.

Hrvat A., Schmidt M., Obholzer M., Benders S., Kollenda S., Horn P.A., Epple M., Brandau S, & Mallmann-Gottschalk N. (2022). Reactivity of NK Cells Against Ovarian Cancer Cells Is Maintained in the Presence of Calcium Phosphate Nanoparticles. Frontiers in Immunology, 13, 830938.

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Isolation of PBMC, NK, and T Cells

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Healthy donor blood collected in trisodium citrate blood collection tubes (Sarstedt) diluted with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco, Life Technologies Limited) in a 1:1 ratio and overlayed on a 1.077 g/mL separation medium (Biocoll, Merck Millipore). Density centrifugation was performed at room temperature (400 g for 30 min) without acceleration and brake. Same methodology was used during the isolation of T cells from patient ascites samples. PBMCs were collected and plastic adherence was performed to deplete monocytes by incubating them in a T175 flask (Sarstedt) at 37 °C and 5% CO₂ for 1 h. For NK isolation, NK MACS Isolation Kit (Miltenyi Biotec) was used according to the manufacturer’s instructions. After isolation 5 U/ml recombinant human IL-15 (50 µg, Immuno Tools) was added to NK cells that were used for functional experiments after incubation overnight. For T cell isolation, CD3 human microbeads (Miltenyi Biotec) were used according to the manufacturer’s instructions. Purity of isolated T cells was routinely tested. In case of overnight resting 5–10 U/ml of recombinant human IL-2 (50 µg, Immuno Tools) was added to T cells.

Hrvat A., Schmidt M., Wagner B., Zwanziger D., Kimmig R., Volbracht L., Brandau S, & Mallmann-Gottschalk N. (2023). Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites. Journal of Experimental & Clinical Cancer Research : CR, 42, 235.

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Purification and activation of NK cells

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Healthy donor blood was collected in trisodium citrate blood collection tubes (Sarstedt). Blood was diluted (1:1 ratio) with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco, Life Technologies Limited) and overlaid on a separation medium (1.077 g/mL, Biocoll, Merck Millipore). Separation by density was done by centrifugation at room temperature (400g for 30 min) without acceleration or brake. Collected PBMCs were incubated in a T175 plastic flask (Sarstedt) at 37°C and 5% CO₂ for 1 h for depleting monocytes. NK isolation was performed using NK MACS Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. After isolation, 5 U/mL recombinant human IL-15 (50 µg, Immuno Tools) was added to NK cells that were used for functional experiments after incubation overnight.

Hrvat A., Benders S., Kimmig R., Brandau S, & Mallmann-Gottschalk N. (2024). Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites. Frontiers in Immunology, 15, 1360615.

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Isolation and Culture of Human NK Cells

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For functional experiments, blood was obtained from healthy volunteer de-identified leukocyte reduction filters (Blood Centers of the Pacific, San Francisco, CA). PBMCs were separated by density gradient centrifugation (Histopaque-1077 Sigma, St. Louis, MO) and were suspended in 10% dimethyl sulfoxide (DMSO, Fisher Scientific, Pittsburgh, PA) and 90% fetal bovine serum (FBS; Omega, Tarzana, CA), and then stored in liquid nitrogen. For NK cell recovery, cryovials of PBMCs were transferred to a 37°C water bath, thawed quickly in RPMI-1640 media (with 20% FBS, warmed to 37°C), and then washed in complete cell culture media (RPMI-1640 with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin; Cell Culture Facility, University of California, San Francisco). Cells were counted and viability was confirmed using a Vi-Cell XR (Beckman Coulter Inc, Brea, CA). Cells were cultured overnight at 37°C with 5% CO₂ in a 24-well plate at a concentration of 3x10⁶/ml of RPMI-1640 media. Exogenous IL-2 was not routinely added to the culture conditions, but only added (1,000 U/ml of IL-2, Biovision, Milpitas, CA) in experiments testing the specific contribution of IL-2. NK cells were isolated from PBMCs using a MACS NK Isolation kit (Miltenyi Biotec Inc., Auburn, CA).

Du J., Lopez-Verges S., Pitcher B.N., Johnson J., Jung S.H., Zhou L., Hsu K., Czuczman M.S., Cheson B., Kaplan L., Lanier L.L, & Venstrom J.M. (2014). CALGB 150905 (Alliance): Rituximab broadens the anti-lymphoma response by activating unlicensed NK cells. Cancer immunology research, 2(9), 878-889.

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Isolation and Activation of Primary CTLs

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Primary CTLs were isolated from the spleens of C57BL/6.OTI, OTI.GzmAB^−/−, or OTI.Prf^−/− mice and activated with OVA₂₅₇ peptide, as described previously (Jenkins et al., 2009 (link)). Cells were Ficoll purified on day 4 and used on day 6 or 7. Mouse NK cells were isolated from spleens by negative selection using the MACS NK isolation kit and autoMACS separator (Miltenyi Biotec). Isolated NK cells were maintained at 7 × 10⁵ cells/ml in RPMI 1640 medium containing 1000 U/ml human IL-2 for 6–8 d before use.

Jenkins M.R., Rudd-Schmidt J.A., Lopez J.A., Ramsbottom K.M., Mannering S.I., Andrews D.M., Voskoboinik I, & Trapani J.A. (2015). Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time. The Journal of Experimental Medicine, 212(3), 307-317.

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Generating Polyclonal NK Cells from AML Patients

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Eighty-four AML patients aged 18–79 years in first CR who were not eligible for allogeneic stem cell transplantation were enrolled in a phase IV clinical trial (Re:Mission). Three patients withdrew consent; the other patients received 10 consecutive 3-week courses of HDC (0.5 mg, subcutaneously twice daily) and low-dose IL-2 (16 400 IU/kg, subcutaneously twice daily) over a period of 18 months. Two patients received allogeneic transplantation after relapse, but died within the follow-up period and were included as events in the OS analysis. Results for primary end points and patient characteristics can be found in references 14 15 34 35 (link). Patient samples were collected before and after the first treatment cycle. Healthy donor peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Lymphoprep (Stemcell Technologies). NK cells were generated by negative selection using MACS NK isolation kit (Miltenyi Biotec). To generate polyclonally activated NK cells, NK cells were cultured with irradiated 221G and allogeneic PBMCs in complete medium supplemented with 600 IU/mL IL-2 (Chiron) and 5 µg/mL phytohemagglutinin (PHA-M, Sigma). After 5 days, the medium was gradually replaced with the same medium without PHA-M.

Hussein B.A., Kristenson L., Pesce S., Wöhr A., Tian Y., Hallner A., Brune M., Hellstrand K., Tang K.W., Bernson E, & Thorén F.B. (2023). NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells. Journal for Immunotherapy of Cancer, 11(8), e007202.

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