A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Summary

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Abstract

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

Introduction

Natural killer (NK) cells are a sophisticated subset of human innate lymphocytes critically involved in the elimination of virally infected cells, transformed cells, and other pathogenic threats ^1,2. NK cell lytic granules house cytotoxic proteins, such as perforin and granzymes. Upon activation, NK cells form a complex interaction with their targets known as immunological synapse, whereby these cytolytic molecules are locally released, resulting in direct target cell lysis and apoptosis, together with cytokine and chemokine release and ultimately in the induction of an inflammatory state ¹

Protocol

Samples were collected according to the ethical guidelines established by the UCLA Human Research Protection Program and IRB approved.

1. Preparation of reagents

NOTE: Unless otherwise stated, all reagents should be allowed to equilibrate at room temperature prior to use. All reagents must remain sterile.

1. Prepare a 2x working solution of Tween-20 (i.e., 0.2%) by adding 10 µL of Tween-20 solution to 5 mL of phosphate-buffered saline (PBS) without calcium and magnesium with a p20 pipette.
   1. Given the high viscosity of Tween-20, take the following steps to ensure accuracy: collect slo....

Results

Before setting up the assay, it is highly recommended that NK cell content be assessed in the effector population of choice. Figure 1 shows a typical CD56 staining before (light blue) and after (red) NK cell enrichment. NK cells comprise up to 15% of PBMCs and should be at least 80% pure after enrichment.

Flow cytometric analysis in this assay involves detection of two parameters: CFSE, detectable i.......

Discussion

The method described here provides a straightforward and cost-effective alternative to the traditional ⁵¹Cr release assay to assess NK cell cytotoxic activity. This method is sensitive, reproducible, and less time-consuming than previous standard methods, like CRA, and can be used for both clinical and research applications.

While the assay works with both total PBMCs and enriched NK cells, the option to use PBMCs without the need to purify cell populations is a great benefit when d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate-buffered Saline (1x, w/o Ca2+ and Mg2+)	Corning (Cellgro)	21-040-CM
Ficoll-Paque PLUS	GE Healthcare	17-1440-02
Tween-20	Sigma	BP337-100
RPMI 1640 Media	Corning (Cellgro)	10-040-CV
Heat-inactivated Fetal Bovine Serum	Omega Scientific	FB-02
Penicillin Streptomycin	Life Technologies	15140-163	Stock solution at 10,000 U/mL
IL-2	R&D Systems	202-IL-050	Lyophilized from a 0.2 μm filtered solution in Acetonitrile and TFA with BSA as a carrier protein. Reconstitute with 500 ul at 100 μg/mL in sterile 100 mM Acetic Acid containing at least 0.1% bovine serum albumin (2.1x10E6 IU/ml)
K562 Cells	ATCC	CCL-243	Cancer cell line
T-75 cell culture flasks	Corning	431464
CFSE cell proliferation kit	Life Technologies (CellTrace)	C34554	Reconstitute I vial with 18 ul DMSO to prepare a 5mM stock solution. Do not freeze/thaw.
Sytox Red	Life Technologies	S34859	Stock solution is provided at 5 μM in 1 mL DMSO. The DMSO solution may be subjected to multiple freeze-thaw cycles without reagent degradation.
Sodium/lithium heparin blood collection tubes	BD	02-687-95
U-bottom 96-well plate	Corning	CLS3897
Serological pipettes	BD Falcon
Polystyrene round-bottom tubes (5mL)	BD Falcon	14959-5
50 mL polypropylene conical tube	BD Falcon	352070
15 mL polypropylene conical tube	BD Falcon	352097
Reagent reservoir	USA Scientific	2321-2230
Human NK cell enrichment cocktail	StemCell Technologies (RosetteSep)	15065