An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

Abstract

Source: Hsiao, C. H., et al. Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment. J. Vis. Exp. (2020).

This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of materials

1. Store reagents, antibodies, and chemicals as shown in the Material Safety Data Sheet (MSDS). Dissolve the drugs or cytokines in solvents as stock solutions and then aliquot for storage at -20 °C or -80 °C.
   NOTE: Detailed information for material preparation is noted in the Table of Materials.

Results

Figure 1: The proportion of CD3⁺CD56⁺ T cells from a representative PBMC sample. (A) Lymphocytes were recognized by specific size and granularity. Selected single cell population for analysis by flow cytometry........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7-Amino Actinomycin D	BD	559925
APC Mouse Anti-Human CD56 antibody	BD	555518	B159
APC Mouse IgG1, κ Isotype Control	BD	555751	MOPC-21
BD FACSCanto II Flow Cytometer	BD	338962		SN: R33896202856
Carboxyfluorescein diacetate succinimidyl ester (CFSE)	BD	565082		Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO
D-(+)-Glucose solution	SIGMA	G8644		For K562 cell culture. Add 12.5 mL to 500 mL of complete medium
Dulbecco's Modified Eagle Medium/F12	HyClone	SH30023.02		Basal medium for OC-3 cell culture
Fetal bovine serum	HyClone	SH30084.03		For K562 and OC-3 cell culture. Complete medium contains 10% of FBS
Ficoll-Paque Plus	GE Healthcare Life Sciences	71101700-EK		Density gradient solution
FITC Mouse Anti-Human CD3 antibody	BD	555332	UCHT1
FITC Mouse IgG1, κ Isotype Control	BD	555748	MOPC-21
Human anti-CD3 mAb	TaKaRa	T210	OKT3	Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 °C
Penicillin-Streptomycin	Gibco	15140122		Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 °C.
Proleukin	NOVARTIS			Reconstitution of Proleukin Powder (22x106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 °C
Recombinant Human Interferon-gamma	CellGenix	1425-050		Reconstitution of rh IFN-g (5x105 IU/50 µg) with 200 µL of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 °C
Recombinant Human Interleukin-1 alpha	PEPROTECH	200-01A		Reconstitution rh IL-1α (10 µg) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 °C
RPMI1640 medium	Gibco	11875-085		Basal medium for K562 cell culture. Storage stock at 4 °C
Sigma 3-18K Centrifuge	Sigma	10295
TrypLE Express Enzyme	Gibco	12605028		Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature
X-VIVO 15 medium	Lonza	04-418Q		Basal medium for PBMC and CIK cells. Storage at 4 °C