Complete blood counts were determined using an automated hematology analyzer in duplicate (Sysmex XN-330, Sysmex Co., Kobe, Japan). Immediately after complete blood counting, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation as previously described (Stem Cell Technologies, Vancouver, Canada) (19 (link)). After isolation, PBMCs were counted using a flow cytometer (BD Accuri C6, Ann Arbor, MI) and an automated cell counter (Countess 3 FL;ThermoFisher Scientific Inc.) Then, according to the manufacturer’s instructions, NK cells were isolated by negative selection using magnetic-activated cell sorting (MACS) separation beads (Miltenyi Biotec, Auburn, CA). Negatively-sorted NK cells were washed at 400g for 10min before resuspending with RPMI-1640. The purity of cells after separation was confirmed using anti-CD3, and anti-CD56 antibodies, to ensure a minimum purity of 90% NK cells were achieved.
Macs separation beads
Manufactured by Miltenyi Biotec
Sourced in Germany
MACS separation beads are a magnetic particle-based technology used for the separation and enrichment of target cells from complex biological samples. The beads are coated with antibodies or other affinity ligands that bind specifically to surface molecules expressed on the target cells. When the sample is exposed to a magnetic field, the labeled target cells are magnetically retained while the unlabeled cells pass through, enabling efficient separation and purification of the desired cell population.
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Lab products found in correlation
2 protocols using macs separation beads
1
Isolation and Purification of NK Cells
2
Isolation and Characterization of Murine Immune Cells
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Cell suspensions from the lymph nodes or the spleens of 6- to 12-week-old mice were stained with specific antibodies (listed in the ‘Reagents’ section) and sorted using a FACS Aria III (BD Biosciences, San Jose, CA, USA) with a typical final purity of >97%. Where required, CD8+ cells and APCs were sorted using magnetic MACS Separation Beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. For flow cytometry, cells were stained with specific antibodies for 30 min on ice after blocking Fc receptors with anti-CD16/CD32. Propidium iodide (PI) solution (Dojindo Laboratories, Kumamoto, Japan) was added at 0.1 µg ml−1 to exclude dead cells. Stained cells were examined using a MACS Quant flow cytometer (Miltenyi Biotec). Live cells were identified as PI− cells with appropriate intensities on FSC and SSC, and further gating was performed as described in the figure legends using FlowJo software (FlowJo LLC, Ashland, OR, USA).
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