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Updated: Aug 9, 2026
Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood
Published on: March 17, 2017
[MTT-colorimetric method for detection the cytotoxic activity of human natural killer cells]
Abstract:
A test system has been adapted for evaluating the cytotoxicity of human natural killer cells (NK) towards tumor targets K-562 in vitro. The method is based on the capacity of mitochondrial enzymes of viable cells to reduce the yellow soluble salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple-blue insoluble formasane precipitate which is quantified spectrophotometrically after dissolution in an organic solvent. The amount of MTT-formasane production is directly proportionate to the count of viable cells. The optimal conditions for the cytotoxicity assay were determined: cell count 10 x 10(3) or 20 x 10(3) cells/well for K-562 as targets and 100 or 200 x 10(3) cells/well for peripheral blood mononuclears as effectors at effector/target cell ratios 20:1, 10:1, and 5:1. This colorimetric method has several advantages over radioactive methods: multiwell scanning spectrophotometers permit the processing of numerous samples and are safe. The results of MTT assay and morphological assay for measuring the NK cytotoxicity were in good correlation and were similarly sensitive.
Insights
This study presents a new in vitro assay for measuring natural killer (NK) cell cytotoxicity against K-562 tumor cells. The MTT assay offers a safe and efficient alternative to radioactive methods for quantifying NK cell activity.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Natural killer (NK) cells are crucial for innate immunity, targeting tumor cells.
- Accurate in vitro assays are needed to evaluate NK cell-mediated cytotoxicity.
- Existing methods, like radioactive assays, have limitations regarding safety and throughput.
Purpose of the Study:
- To adapt and validate a colorimetric MTT assay for quantifying human NK cell cytotoxicity against K-562 tumor cells.
- To determine optimal assay conditions for reliable and sensitive NK cell activity measurement.
- To compare the MTT assay with a morphological assay for NK cytotoxicity assessment.
Main Methods:
- Utilized the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, which measures mitochondrial enzyme activity in viable cells.
- Optimized effector (peripheral blood mononuclear cells) to target (K-562) cell ratios and cell counts.
- Quantified formazan precipitate spectrophotometrically after dissolution in an organic solvent.
Main Results:
- Established optimal conditions for NK cell cytotoxicity assay using specific cell counts and effector/target ratios (20:1, 10:1, 5:1).
- Demonstrated that MTT formazan production is directly proportional to the number of viable cells.
- Showed good correlation and comparable sensitivity between the MTT assay and a morphological assay for NK cytotoxicity.
Conclusions:
- The MTT assay is a reliable, safe, and efficient colorimetric method for evaluating NK cell cytotoxicity in vitro.
- This method allows for high-throughput analysis using multiwell scanning spectrophotometers.
- The MTT assay provides a sensitive and reproducible alternative to traditional cytotoxicity assays.
