Application protocol

Fast isolation of NK cells from human whole blood

This application protocol is based on a scientific poster that can be accessed under "Resources" in the Protocol tab.

 In this application protocol, we describe an easy and convenient technique using MACSxpress® Technology for the efficient purification of untouched immune cells from human whole blood in 20 minutes. Up to 30 mL of whole blood can be processed in a single tube, enabling the fast isolation of a large number of NK cells for downstream applications. The procedure does not require density gradient centrifugation, which minimizes pipetting steps and aerosol formation for a high level of safety.

Protocol

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

Materials

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

General reagent and instrument requirements

  • Buffer: Phosphate-buffered saline (PBS) pH 7.2, supplemented with 0.5% human serum albumin (HSA) and 2 mM EDTA. Keep buffer cold (2–8 °C). Degas buffer before use, as air bubbles could block the column. 
    Note:  HSA can be replaced by other proteins such as bovine serum albumin, fetal calf serum or human AB serum. Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
  • Medium: NK MACS® Medium (# 130-107-879) supplemented with 5% AB serum and 500 IU/mL human Interleukin 2 (IL-2)
  • Human IL-2, e.g., Human IL-2 IS, premium grade (# 130-097- 748)
  • (Optional) MACS GMP Cell Expansion Bags (# 170-076-403) or flat bottom cell culture plates with lids
  • Humidified incubator
  • MACSmix™ Tube Rotator (# 130-090-753)
  • MACSQuant® Analyzer (# 130-092-197)
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometric analysis, e.g., CD45-VioBlue® (# 130-092-880), CD3-FITC (# 130-080-401), and CD56-PE (# 130-090-755). Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells.

For fast isolation of NK cells from whole blood

  • MACSxpress® Whole Blood NK Cell Isolation Kit, human (#  130-098-185)
  • MACSxpress Separator (# 130-098-308)
  • 5 mL polystyrene round-bottom test tube or 15 mL or 50 mL tubes
  • (Optional) MACSxpress Erythrocyte Depletion Reagent (# 130‑098-196)
  • (Optional) Red Blood Cell Lysis Solution (10×) (# 130-094-183)

For activation and expansion of NK cells

  • NK Cell Activation/Expansion Kit, human (# 130-094-483)

For cytotoxicity assay

  • K562 target cells
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