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

Published: November 30, 2023

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 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.<br…

Representative 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. (B) Statistical analysis of CIK expansion efficacy from three healthy donors was conducted using a t-test (*, p < 0.01). <p class="jove_conten…

Disclosures

The authors have nothing to disclose.

Materials

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 (22×106 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 (5×105 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

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Cite This Article
An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells. J. Vis. Exp. (Pending Publication), e21802, doi: (2023).

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