Home › Immunology › Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Immunology JoVE (Open Access) Citable · DOI

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

DOI: 10.3791/2540-v
What you'll learn
  • ✓ Isolate and purify human NK cells from peripheral blood using density gradient separation
  • ✓ Expand NK cells in culture using cytokine stimulation and feeder cells
  • ✓ Perform and interpret NK cell cytotoxicity assays to assess functional killing capacity
Protocol

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Difficulty
intermediate
Total time
~10–14 days (including 7–10 day expansion phase)
Model organism
Human peripheral blood mononuclear cells (PBMCs)
Biosafety
BSL-2

Steps

1
Isolate cell fractions from buffy coat

Use density gradient centrifugation to separate peripheral blood mononuclear cells (PBMCs) from buffy coat material. Collect the intermediate cell layer for downstream NK cell processing.

▶ 01:40
2
Expand isolated NK cells in culture

Culture purified NK cells with cytokine stimulation and feeder cells to generate large numbers of functional NK cells over 7–10 days.

▶ 03:34
3
Assess NK cell cytotoxicity against target cells

Perform a functional killing assay by co-culturing expanded NK cells with target cells and measuring cytotoxicity through established readouts (e.g., chromium release or fluorescence-based assays).

▶ 06:34
4
Analyze and interpret representative results

Review cell expansion kinetics, purity metrics, and cytotoxicity data to confirm successful NK cell isolation, expansion, and functional competence.

▶ 08:33
💬 Comments coming soon