Application protocol

NK cells expansion from human PBMCs or isolated NK cells

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.


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step. 


The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For isolation of PBMCs

Isolation of PBMCs using Ficoll-Paque™

  • 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).
  • Ficoll-Paque (ρ = 1.077 g/mL)
  • 50 mL conical centrifuge tubes  

For isolation of NK cells

  • NK Cell Isolation Kit, human (# 130-092-657)
  • PBE 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.
  • (Optional) Pre-Separation Filters (30 μm) (# 130-041-407) to remove cell clumps
  • MACS MultiStand (# 130-042-303)
  • Choose the appropriate MACS Separator and MACS Columns:
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
MS1×1072×108MiniMACS™, OctoMACS™
LS1×1082×109MidiMACS™, QuadroMACS™
autoMACS2×1084×109autoMACS Pro

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.

For NK cell expansion


  • NK MACS Medium (# 130-114-429)
  • Human IL-2 IS, premium grade (e.g. # 130-097-744)
  • Human AB serum or autologous plasma. Availability is country-specific.
  • 24-well, 12-well, and 6-well cell culture plates
  • (Optional) T25, T75, and T175 flasks

For flow cytometry analysis

  • CD3 Antibody, anti-human, APC, REAfinity™ (# 130-113-135)
  • CD56 Antibody, anti-human, PE, REAfinity (# 130-113-312)
  • CD45 Antibody, anti-human, VioBlue®, REAfinity (# 130-110-637)
  • CD19 Antibody, anti-human, APC-Vio® 770, REAfinity (# 130-113-643)
  • CD14 Antibody, anti-human, FITC, REAfinity (# 130-110-518)
  • 7-AAD Staining Solution (# 130-111-568)
  • (Optional) CD16 Antibody, anti-human, PE-Vio 615, REAfinity (# 130-107-710)
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