Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis
Published: November 30, 2023
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 Transfer the following into separate and appropriately labeled 1.5 ml tubes: Add 500 µL of fresh IMDM containing resuspended DiO-labeled K562 cells into the "Double Positive" labeled 1.5 mL tube. Add 500 µL of fresh IMDM containing resuspended DiO-labeled K562 cells into the "DiO only" labeled 1.5 mL tube. Add 500 µL of fresh IMDM containing resuspended unlabeled K562 cells into the "Propidium…
Representative 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 made using the image attached to each event. This can be accessed in analysis software by simply clicking on the event on the graphs. (…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis. J. Vis. Exp. (Pending Publication), e21846, doi: (2023).