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Single Tube HT Cytotoxicity Assays

Cytolytic activity is an important process for eliminating intracellular pathogens and cancerous cells. This process is accomplished through various immune effector mechanisms including Natural Killer (NK) leukocytes. Natural killer activity is accomplished by non-specifically lysing infected targets through the use NK receptors or the FcgII (CD16) receptor recognizing IgG bound to specific antigens on the target cell surface (1). 

The assessment of NK cytolytic activity has been previously analyzed by detecting lactate dehydrogenase release or more commonly, through the detection of radioactive chromium-51 (51Cr), which is released from lysed target cells (1). These techniques however have several drawbacks such as: high spontaneous leakage resulting in high backgrounds, short half-life, high cost, and the potential health risk associated with the use of radioactive materials (2). 

Flow cytometric assays have been developed to overcome some of the difficulties associated with lactate dehydrogenase and 51Cr release assays. Radosevic et al (1990) reported the detection of NK cytotoxicity activity by staining the target cells with the green fluorescent dye F-18, in combination with the DNA intercalating dye, propidium iodide (3). In addition, a red fluorescent membrane dye, PKH-26, has been used in preference to F-18 and in combination with the viability probe, TO-PRO-3 iodide (4-7). The use of PKH-26 can also be problematic as it is difficult to use at a constant concentration due to unreliable staining, and the staining procedure requires multiple steps, often decreasing the viability of the target cells. The problems associated with cell staining were overcome when Olin et al (2004) reported on the use of (5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) membrane stain for use as a target cell labeling stain (8). By staining K562 cells with CFSE, an increase in NK activity following BCG vaccination was demonstrated.   Using the same technique in staining Mycobacterium infected monocytes, they further demonstrated specific antigen directed cytolytic activity against Mycobacterium.

Following the optimization of the flow cytometer assay, Lee-MacAry (2001) compared a flow cytometric and 51Cr release assay, demonstrating a correlation greater than 95% (1). The strengths of the flow cytometric based assay are the elimination of radioactive materials and the ability to analyze cytolytic activity at a single cell level. Recently, International Qualex Flow Cytometry Systems has modified the flow cytometric assay, overcoming the problems previously associated with flow cytometric cytolytic assays. This flow cytometric cytolytic assay kit contains enough reagents for 125 or 250 test assays and optimization of experimental design including the establishment of optimal effector/target cell ratios.

Assay Principle

The fluorescent capability of the flow cytometer allows the membrane stain to separate the target and effector cell populations for analysis. This is accomplished by labeling the target cells with the green fluorescent CFSE membrane stain. Following the labeling of the target cells, effector cells are then added to the stained target cell population at a pre-determined effector/target cell ratio, as defined by each individual investigator, and incubated together in a 0.4 mL (400 mL) volume of sterile cell culture media for a previously optimized (effector + target cell) incubation period. Upon completion of the effector + target cell incubation period, the live/dead stain is added to measure the percentage of cell death, or cytotoxicity.


Figure 1. Cytolytic activity was determined by a shift up the Y-axis from R1 to R2 regions due to an increase of dead or dying K562 cells. By adjusting effector/target cell ratios, a linear response is observed during the optimization steps of the cytolytic assay.

In addition, IQFCS has optimized the total cytotoxicity assay to distinguish apoptosic from necrotic cells resulting in the most sensitive cytolytic assay available. The total cytotoxicity kits not only are sensitive, this kit allows researchers to determine mechanisms involved in cell killing.

Total Cytotoxicity assay

Cell Calculation Spreadsheet

Cytotoxicity assay kits available:


Cytotoxicity Manual


1. Lee-MacAry, A.E., Ross, E.L, Davies, D., and Wilkinson, R.W., 2001. Development of a novel flow cytometric cell-mediated cytotoxcity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J. Immunol. Methods 252: 83-92.

2. Radosevic, K., Garritsen, H.S.P., Van Graft, M., De Grooth, B.G. and Greve, J., 1990. A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells. J. Immunol. Methods 135:81

3. Hatam, L,. Schuval, S., Bonagura, V.R., 1994. Flow cytometric analysis

of natural killer cell function as a clinical assay. Cytometry 16:59.

4. Lowdell, M.W., Ray, N., Graston, R., Corbett, T., Deane, M. and Prentice, H.G., 1997. The in vitro detection of anti-leukemia-specific cytotoxicity after autologous bone marrow transplantation for acute leukemia. Bone Marrow Transplant. 19:891.

5. Van Hooijonk, C.A.E.M., Glade, C.P. and Van Erp, P.E.J., 1994. TO-PRO-3 Iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. Cytometry 17:185.

6. O’Brien, M.C. and Bolton, W.E., 1995. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19:243.

7. Olin, M., K. Choi, J. Lee, and T. W. Molitor. 2004. Gamma Delta T-lymphocytes cytolytic activity against Mycobacterium tuberculosis analyzed by flow cytometry. J Immunol Methods, 297:1-11..

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