Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Abstract

Source: McBride, J. E., et al. Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood. J. Vis. Exp. (2017).

This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

Protocol

1. Preparation of Controls

1. Transfer the following into separate and appropriately labeled 1.5 ml tubes:
   1. Add 500 µL of fresh IMDM containing resuspended DiO-labeled K562 cells into the "Double Positive" labeled 1.5 mL tube.
   2. Add 500 µL of fresh IMDM containing resuspended DiO-labeled K562 cells into the "DiO only" labeled 1.5 mL tube.
   3. Add 500 µL of fresh IMDM containing resuspended unlabeled K562 cells into the "Propidium Iodide (PI) only" labeled 1.5 mL tube.
2. Place the Double Positive and PI-only tubes in a 55 °C water bath for 5 min.
3. After the 5 min has....

Results

Figure 1: Representative histograms, scatter plots, and images for cytotoxic activity analysis. (A) focus cell analysis. (B) single cell analysis. (C) target cell staining analysis. All determinations are m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
K-562 lymphoblasts	ATCC	CCL-243
Iscove's Modified Dulbecco's Media	ATCC	30-2005	High glucose, with L-Glutamine, with HEPES, Sterile-filtered
Alpha Minimum Essential medium	ATCC	CRL-2407	Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate
Propridium Iodide Staining Solution	BD Pharmingen	51-66211E
Vybranto DiO cell-labeling solution	Vybranto	V-22886
ImageStream X Mark II Imaging Flow Cytometer
Whole Blood CD56 MicroBeads, human	Miltenyi Biotec	130-090-875
ImageStream X Mark II Imaging Flow Cytometer	EMD Millipore
Speedbeads	Amnis Corporation	400030
INSPIRE Software	EMD Millipore	Version Mark II, September 2013
Ideas Application Software	EMD Millipore		Version 6.1, July 2014