Spleen and liver tissues were cut into small pieces with a sterile scalpel and passed through 40-μm mesh filters. Single-cell suspensions of splenocytes were prepared as previously described [6 (link),18 (link)]. Liver mononuclear cells (LMNCs) were enriched by density-gradient centrifugation as previously described [19 (link)–21 ]. Murine NK cells were isolated from splenocytes and LMNCs by negative selection using the MACS NK cell isolation kit II (Miltenyi Biotec). The purity of unlabeled NK cells was ~85%, as determined by flow cytometry. The activation status of NK cells was not affected by the negative selection process. Since the transferred cells contained ~15% cells other than NK cells, we depleted contaminating CD4+T cells in recipient Rag2−/−Il2rg−/− mice by injection of anti-CD4 mAb (200 ug/mouse) (GK1.5, Biolegend) on -1, 0, and 3 DPI.
Macs nk cell isolation kit 2
Manufactured by Miltenyi Biotec
Sourced in Germany
The MACS NK cell isolation kit II is a laboratory product designed for the isolation of natural killer (NK) cells from various sample types. The kit utilizes magnetic separation technology to selectively enrich NK cells from cell suspensions.
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Lab products found in correlation
2 protocols using macs nk cell isolation kit 2
1
Murine NK Cell Isolation and Purification
2
Isolation of NK Cells from Peripheral Blood
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The fresh heparinized peripheral blood was obtained from totally 10 healthy subjects with no history of leishmaniasis after receiving their informed consent. Donors were not from endemic area and verified by leishmanin test previously. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood on a Ficoll gradient (Sigma, Missouri, USA), as described previously (14 (link)). NK cells were isolated by negative selection using the MACS NK cell isolation kit II (Miltenyi Biotech, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions. Briefly, PBMCs were washed twice, resulted pellet was resuspended in 40 μl of MACS buffer per 107 total cells, and 10 μl of NK cell biotin-antibody cocktail was added. After incubation for 5 minutes in the refrigerator (2–8 °C), 30 μl MACS buffer and 20 μl NK cell microbead cocktail were added per 107 total cells. Cells were incubated for an additional 10 minutes in the refrigerator and the volume was adjusted to a minimum of 500 μl of buffer. Finally, magnetic separation was done with the autoMACS separator.
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