How to Detect ADCC

Chromium 51 (51Cr) Labeling/Release

The isotope 51Cr is the most classical method to evaluate the cytotoxicity assay of immune cell killing of tumor cells. Target cells are specifically labeled with 51Cr, washed, and centrifuged to remove excess 51Cr, and then effector cells are added for co-culture. After the effector cells kill the target cells, the target cells are lysed and 51Cr is released into the supernatant, and then the 51Cr supernatant is detected using a Gamma counter or a flash counter to evaluate the killing activity.

Target Cell Labeling

This method utilizes flow cytometry to quantify the ADCC killing target cells. In detail, target cells are labeled with CFSE, and dead cells are labeled with FVD (which has higher stability than commonly used 7-ADD and PI dyes). CFSE⁺FVD⁺ double positive is the dead target cell, which can be used to quantify the target cells killed by ADCC.¹

Fig.1 CFSE⁺FVD⁺ characterizes the ADCC effect.¹

Fluorescent Labeling

This is a method that fluorescently labels live cells. In detail, it introduces an acetylmethoxymethyl ester group on the basis of calcein, which increases hydrophobicity and allows it to freely penetrate the cell membrane. Calcein-acetylmethoxymethyl itself is not fluorescent, penetrates the cell membrane, and is cleaved by intracellular esterases to form the polar molecule calcein, which loses its membrane-free permeability and emits strong green fluorescence.

Another fluorescent labeling method is similar to the 51Cr labeling/release method, which uses the hydrophobic ligand BATDA to label the target cells, and the esterified BATDA freely penetrates the cell membrane and is deacetylated by intracellular acetysterase to produce the hydrophilic ligand TDA, which loses the free permeability of the membrane and remains in the cell. After BATDA labels the target cells, wash and centrifuge to remove the excess dye, and then add effector cells for co-culture, the lysed target cells release TDA into the europium (Eu)-containing supernatant, and form a highly fluorescent and stable Eu-TDA complex with Eu, and the lysis of the target cells can be evaluated by detecting the time-resolved fluorescence signal of the complex.

LDH Release

This method can be used as an important indicator of cell integrity. When apoptosis or necrosis, the destruction of cell membrane structure causes the release of enzymes in the cytoplasm into the culture medium, including LDH with relatively stable enzyme activity. Cytotoxicity can be quantified by detecting the activity of LDH in the medium. The principle of LDH detection is as follows: in the process of LDH catalyzing the formation of pyruvate from lactic acid, the oxidized coenzyme (NAD+) becomes reduced coenzyme (NADH), and NADH is catalyzed by lipoamide dehydrogenase (diaphorase) in NADH and INT to generate NAD⁺, and the absorption peak is generated at 490 nm wavelength, so that the activity of lactate dehydrogenase can be quantified by colorimetry. Absorbance is linearly positively correlated with lactate dehydrogenase activity.

Fig.2 ADCC assay by LDH.²

References

1. Yamashita, M.; et al. "A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells." Scientific reports. (2016). 6, 19772.
2. Khanna, Vidhi.; et al. "Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC)." Scientific reports. (2021) 11,1 3346.