CD Marker Recognition of Human Cytokine-Induced Killer Cells by Flow Cytometry
Published: August 31, 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)
In this video, we demonstrate a flow cytometry-based technique to determine the proportion of cytokine-induced killer T-cells, CIK cells, expanded from a peripheral blood mononuclear cell culture.
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. CIK induction and expansion On Day 0, culture the PBMCs (1 x 106) in fresh basal medium containing 1,000 IU/mL of IFN-γ for 24 h in a humidified cell culture incubator at 37 °C and 5% CO2. On Day 1, re…
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).
Declarações
The authors have nothing to disclose.
Materials
| 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 |
| 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 |
| RPMI1640 medium | Gibco | 11875-085 | Basal medium for K562 cell culture. Storage stock at 4 °C |
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CD Marker Recognition of Human Cytokine-Induced Killer Cells by Flow Cytometry. J. Vis. Exp. (Pending Publication), e21559, doi: (2023).