CD Marker Recognition of Human Cytokine-Induced Killer Cells by Flow Cytometry

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

1. 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.
2. On Day 1, refresh the medium with fresh basal medium containing 50 ng/mL of anti-CD3 antibody, 1 ng/mL of rh IL-1α, and 1,000 U/mL of rh IL-2. Refresh the me....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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<br/> Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 &deg;C
RPMI1640 medium	Gibco	11875-085	Basal medium for K562 cell culture. Storage stock at 4 &deg;C