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