Nk macs supplement

Manufactured by Miltenyi Biotec

Sourced in Germany

NK MACS supplement is a cell culture medium supplement designed to support the growth and expansion of natural killer (NK) cells. It contains a proprietary blend of cytokines and other growth factors that are essential for the activation and proliferation of NK cells.

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Lab products found in correlation

Nk macs medium, by Miltenyi Biotec (3 mentions) Human ab serum, by Merck Group (2 mentions) Human il 2, by Miltenyi Biotec (1 mentions) Rosettesep nk cell enrichment mixture, by STEMCELL (1 mentions) Recombinant human il 2, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Nk cell isolation kit, by STEMCELL (1 mentions) E6130, by MedChemExpress (1 mentions) Click s, by Irvine Scientific (1 mentions) Vectofusin 1, by Miltenyi Biotec (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Il 15, by Immunotools (1 mentions) Il 12, by Miltenyi Biotec (1 mentions)

6 protocols using nk macs supplement

Expansion of Human NK Cells

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Leukoreduction system (LRS) chambers
from different donors were sourced from Stanford Blood Center. Peripheral
blood mononucleated cells were isolated by density gradient. Human
PBMC cells at 0.8 × 10⁶/mL were cultured in NK MACS
medium supplemented with 5% human AB serum (Millipore Sigma), NK MACS
supplement, and 500 IU/mL human IL-2 (Miltenyi Biotec). Human NK cells
were cultivated and expanded for 10–14 days. The CD3^–CD56⁺ NK cells were confirmed by FACS staining.

Ren J., Jo Y., Picton L.K., Su L.L., Raulet D.H, & Garcia K.C. (2022). Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism. ACS Synthetic Biology, 11(10), 3426-3439.

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Isolation and Activation of Human NK Cells

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Human NK-cells were isolated from PBMC of healthy donors with the RosetteSep NK-cell enrichment mixture method (Stem-Cell Technologies, IT). NK-cells with purity greater than 90% were stimulated with 100 IU/mL of recombinant human IL2 (PeproTech, FR) for 48 hours at 37°C. NK-cells were maintained in culture with NK MACS medium supplemented with 5% human serum and 1% NK MACS supplement (Miltenyi Biotech, DE).

Caforio M., Tumino N., Sorino C., Manni I., Di Giovenale S., Piaggio G., Iezzi S., Strimpakos G., Mattei E., Moretta L., Fanciulli M., Vacca P., Locatelli F, & Folgiero V. (2023). AATF/Che-1 RNA polymerase II binding protein overexpression reduces the anti-tumor NK-cell cytotoxicity through activating receptors modulation. Frontiers in Immunology, 14, 1191908.

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Expansion and Chemotaxis of NK Cells

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PBMC were isolated from healthy donors by density gradient centrifugation. Natural killer (NK) cells were purified from PBMC by magnetic bead separation using an NK cell isolation kit as per manufacturers protocol (STEMCELL Technologies, Canada). NK cells were expanded in NK MACS Medium (Miltenyi Biotec, Germany), supplemented with 1% of NK MACS Supplement (Miltenyi Biotec, Germany), 5% Human AB serum (Sigma, USA), 500 IU/ml IL-2 (Peprotech, UK). Manufacturers protocols for expansion were used. For chemotaxis experiments, NK cells were resuspended at a concentration of 1 × 10⁶ cells per 50 µl of MACS media. Cells were left untreated or treated with 5 nM of E6130 (Medchem express, USA) for 1 h prior to assay commencement.

Mylod E., O’Connell F., Donlon N.E., Davern M., Marion C., Butler C., Reynolds J.V., Lysaght J, & Conroy M.J. (2024). Real-time ex vivo monitoring of NK cell migration toward obesity-associated oesophageal adenocarcinoma following modulation of CX3CR1. Scientific Reports, 14, 4017.

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Expansion and Transduction of T Cells and CAR-NK Cells

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T cells were obtained from buffy coats by Ficoll and magnetic T cell depletion (Miltenyi Biotec). T cells were expanded in Click’s media (50% RPMI, 50% Click’s (Irvine Scientific), 5% human serum, 1% Pen/Strep), activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) and IL-2 (100 IU/mL) every other day. Experiments were performed after 8–10 days of T cell expansion. Viral transduction was performed after 48 hours of the expansion at a MOI of 10. NK cells were selected from CB units by Ficoll and magnetic NK cell depletion (Miltenyi Biotec). To produce CAR-NK (ARI3 cells), NK cells from a CB unit were expanded with NK MACS Medium (Miltenyi Biotec), 1% NK MACS Supplement, 5% human serum, IL-2 (500 IU/mL) and IL-15 (140 IU/mL). On day 5, CB-NK were transduced at a multiplicity of infection (MOI) of 5 with Vectofusin-1 (10 mg/mL) following Vectofusin-1 protocol (Miltenyi Biotec). Cells were washed 6–24 hours after transduction. On day 7, ARI3 cells were coexpanded with feeder cells adding IL2 (400 IU/mL) every other day for the next 7 days. Of note, throughout the manuscript, ‘ARI3 cells’ refers to CAR-NK cells and CB-NK refers to untransduced NK cells.

Bachiller M., Perez-Amill L., Battram A.M., Carné S.C., Najjar A., Verhoeyen E., Juan M., Urbano-Ispizua A, & Martin-Antonio B. (2021). NK cells enhance CAR-T cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cell fitness. Journal for Immunotherapy of Cancer, 9(8), e002866.

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Large-Scale Expansion of NK Cells for Serial Killing Assays

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To perform an in vitro serial killing assay in the presence of NK cells (see below), a large-scale activation and expansion of NK cells were set up according to the following method. After thawing, cryopreserved purified human CD56⁺ NK cells, negatively selected (AllCells) (5 × 10⁶ cells) were resuspended at 1 × 10⁶ cells/mL in expansion NK MACS medium (NK MACS medium supplemented with 1% NK MACS supplement (Miltenyi Biotec), 5% human AB serum, IL-2 (500 IU/mL), and IL-15 (140 IU/mL)). Cells were then cultured in a 24-well plate (700 μL/well) and incubated at 37 °C, 5% CO₂ undisturbed for the first 5–6 days. At day 5 or 6, 300 μL expansion NK MACS medium was added without disturbing the cells. On day 7, a fresh expansion NK MACS medium was added to cells to dilute to a final concentration of 5 × 10⁵ cells/mL and cells were cultured in a six-well plate (2.5 mL/well). Starting on day 10, a fresh expansion NK MACS medium was added every 2 days to dilute cells to a final concentration of 5 × 10⁵ cells/mL and cells were cultured in T75 flasks (>7 mL/flask) or T175 flasks (>30 mL/flask). Expanded cells were utilized in serial killing assays starting at day 13 or 14.

Jo S., Das S., Williams A., Chretien A.S., Pagliardini T., Le Roy A., Fernandez J.P., Le Clerre D., Jahangiri B., Chion-Sotinel I., Rozlan S., Dessez E., Gouble A., Dusséaux M., Galetto R., Duclert A., Marcenaro E., Devillier R., Olive D., Duchateau P., Poirot L, & Valton J. (2022). Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Nature Communications, 13, 3453.

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Expansion of Cryopreserved UCB CD34+ NK Cells

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Cryopreserved UCB CD34+ progenitor-derived HPC-NK cells from different donors were kindly provided by Dr. Harry Dolstra (Radboudumc, Nijmegen)( Van der Meer et al., 2021) . The cells were thawed in medium with 71% Human Serum (HS; Sanquin), 0.03% DNAse and 0.1%MgCL 2 and were washed (at 300 g for 15 minutes) after 10 minutes resting. The cells were then resuspended in NK MACS medium (Miltenyi, supplemented with 10% HS and 1% NK MACS supplement (Miltenyi) at the concentration of 3 million cells/mL. 1.5 mL of the cell suspension was loaded into the 6 well plate and were supplemented by 50 ng/mL IL15 (Immunotools, Catalog no. 352310) and 0.2 ng/mL IL12 (Miltenyi, . On every second day, 1 mL NK MACS medium (with 10%HS, 1% NK MACS supplement, 50 ng IL15 and 0.2 ng/mL IL12) was added to the cells, thus allowing them to expand for 7 days. All the assays were performed after the 6 th day of expansion. The culture was kept for 2 weeks after thawing.

Subedi N., Verhagen L.P., Jonge P.K.d., Eyndhoven L.C.V., Turnhout M.C.v., Koomen V., Baudry J., Eyer K., Dolstra H., & Subedi N. (2022). Single cell profiling reveals functional heterogeneity and serial killing in human peripheral and ex vivo-generated CD34+ progenitor derived Natural Killer cells.

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