Many downstream applications require expansion of NK cells prior to analysis. In vitro cultivation of NK cells often results in low expansion rates, exhausted phenotype due to long-term expansion, and overgrowth of conventional T cells if present in the initial culture. NK MACS Medium has a defined formulation that enables expansion of NK cells from peripheral blood mononuclear cells (PBMCs) or isolated NK cells. Expanded cells are fully functional and ready for downstream applications.
PE buffer: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
▲Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
Isolate pure and fully functional NK cells using the NK Cell Isolation Kit, human. This indirect magnetic labeling system isolates untouched NK cells from human PBMCs by magnetically labeling and depleting non-NK cells (i.e., T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes, and erythroid cells) with a cocktail of biotin-conjugated antibodies and the NK Cell MicroBead Cocktail. Follow the protocol in the kit data sheet.
Download the data sheet
PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Degas buffer before use, as air bubbles could block the column.
After staining the samples, perform the flow cytometry analysis as follows:
|Marker on y-axis||Marker on x-axis||Identified cell population|
|CD14||CD56||CD14+ and CD14+CD56+ (monocytes)|
|CD3||CD19||CD3–CD19+ (B cells)|
|CD3||CD56||CD3–CD56+ (NK cells)|
|CD3||CD14||CD3+CD14– (T cells)|
|CD3||CD56||CD3+CD56+ (NKT cells)|
Ex vivo cultivation is an attractive option to increase NK cell numbers for functional analyses, including anti-tumor potential. Miltenyi Biotec NK MACS Medium addresses the major challenges of culturing NK cells with a uniquely designed formulation that expands fully functional NK cells from either PBMCs or isolated NK cells. The medium is xeno-free and enables superior NK cell expansion with minimal growth of unwanted cells, like B, T, or dendritic cells.
▲Note: Supplementation with serum or autologous plasma is necessary.
When adding expansion NK MACS Medium to culture plates or flasks, please note:
Do not disturb cells.
Add 300 µL expansion NK MACS Medium.
Take a 50 µL sample of cells to determine NK cell count and perform cell analysis (e.g., proceed to "Cytotoxicity assay").
NK cells cultivated in NK MACS Medium retain their natural cytotoxicity against the K-562 cell line and are suitable for any downstream applications, including cytotoxicity assays and flow cytometry analysis.
|Column||Max. number of labeled cells||Max. number of total cells||Separator|
▲ Note: When using the NK Cell Isolation Kit the unwanted cell fraction is labeled and the target cells remain unlabeled. Depending on the target cell frequency, the labeled fraction can therefore represent the majority of the total cells. To avoid blocking of the column, do not exceed the max. number of labeled cells per column. Estimate the number of labeled cells in the sample, split the sample if necessary, and use the appropriate number of separation columns.
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