Cell-Mediated Cytotoxicity Assays

Abstract

Cell-mediated cytotoxicity measurements can be divided in two methods, depending on whether radioactive material is used or not. Natural Killer cell-mediated cytotoxicity is routinely measured with a short-term assay by labeling the target cells with radioactive chromium (51Cr). The advantages of this method are (1) highly sensitivity, (2) easy execution, (3) low spontaneous release, and (4) nontoxic markers. The disadvantages include short half-life of the label and handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/enzymatic assays are highly sensitive and easy to use but unfortunately works only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of the dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false positive results leading to a decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.

A Mutated Prostatic Acid Phosphatase (PAP) Peptide-Based Vaccine Induces PAP-Specific CD8+ T Cells with Ex Vivo Cytotoxic Capacities in HHDII/DR1 Transgenic Mice

“…2.8. 51 Cr Release Cytotoxicity Assay ILL-stimulated splenocytes were co-incubated with 51 Cr-labeled [19] T2 target cells in 96-well round-bottom plates at effector: target ratios of 100:1 (500,000:5000), 50:1 (250,000:5000), 25:1 (125,000:5000), and 12.5:1 (62,500:5000)). For 51 Cr labeling, 2 × 10 6 T2 cells were incubated with 1.85 MBq of 51 Cr and 50 µg/mL of ILL 9 mer peptide for 1 h at 37 • C. Following 4 h at 37 • C, 50 µL of the supernatant of each well was transferred to Luma plates (PerkinElmer LAS (UK) Limited, Llantrisant, UK) and left to dry until reading on a Top Count microplate scintillation counter.…”