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. (…

開示

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

Play Video

記事を引用
Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis. J. Vis. Exp. (Pending Publication), e21846, doi: (2023).

View Video