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Total Cytotoxicity



Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells. This process is accomplished through various immune effector mechanisms including natural killer (NK) leukocytes. NK activity is accomplished by non-specifically lysing infected targets through the use of NK receptors, or the FcgII (CD16) receptor, recognizing IgG bound to specific antigens on the target cell surface (1). NK cells may also induce apoptosis in target cells. The activity of natural killer cells, and their effect on target cells, is frequently studied in immunomodulation experiments. 

Older methods to assess NK cytolytic activity include measuring the release of lactate dehydrogenase, and more commonly, the release of radioactive chromium-51 (51Cr) from lysed target cells (1). However, these techniques have several drawbacks such as: high spontaneous leakage resulting in high backgrounds; high cost; short half-life; the potential health risk due to radioactive material (2); and the inability to detect early-stage apoptotic cells.

Flow cytometric assays have been developed to overcome some of the difficulties with lactate dehydrogenase and 51Cr release assays. Radosevic (1990) detected NK cytotoxicity activity by staining target cells with the green fluorescent dye, F-18, in combination with the DNA intercalating dye, propidium iodide (3). Since then, 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), but the PKH-26 method is still problematic. It is difficult to use at a constant concentration, leading to unreliable staining, and the staining procedure requires multiple steps, often decreasing the viability of the target cells. Despite this, following the optimization of a flow cytometric assay, Lee-MacAry (2001) compared it with the 51Cr release assay and demonstrated a correlation greater than 95% (1). 

Since then, the problems with older flow cytometric assays were overcome when Olin (2005) used 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to stain the membranes of target cells (8). By staining K562 cells with CFSE, they demonstrated an increase in NK activity following BCG vaccination. Using the same technique to stain Mycobacterium infected monocytes, they further demonstrated specific antigen-directed cytolytic activity against Mycobacterium.

Building upon the techniques of Olin (2005), International Qualex Flow Cytometry System (IQFCS) has improved the flow cytometric assay by combining it with IQFCS apoptosis detection reagent to concurrently quantify caspase-positive cells as well. Now, in a single tube, scientists can differentiate cytolytic killer activity from apoptosis with IQFCS Total Cytotoxicity Kit. There is no need to lyse the cells or wait for enzyme release (like LDH, ATP, AK and other assays). Since this kit does not use any radioisotopes, it is much safer to run than the 51Cr assays. At least 10% more cytolytic activity is often measured when apoptotic cells are also identified.

IQFCS Total Cytotoxicity Kit includes three fluorescent reagents, which enable the flow cytometer to easily separate the target and effector cell populations for analysis. The first of these reagents, CFSE, a green membrane stain, is used to label all the target cells green. The unstained effector cells are then added and incubated with the target cells.

Apoptotic target cells can then be identified by labeling with the second reagent, an orange-red fluorescent poly caspases inhibitor which binds to active caspase enzymes up-regulated for apoptosis (9). Upon completion of the desired incubation period (which includes exposure to the apoptosis detection probe), the last reagent, 7-aminoactinomycin D (7-AAD), a red live/dead stain, is added to stain all dead cells red by binding to the DNA of membrane-compromised cells.  

Because 7-AAD will not detect cells in the early stages of apoptosis, it is important to expose the target cells to the apoptosis probe. This test will often reveal a significant percentage of cells that were 7-AAD-negative (indicating they are viable) yet caspase positive (apoptotic and dying). These cells cannot be detected by any other method. Careful gating of apoptosis vs 7-AAD distinguishes mechanism of killing 


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Total cytolytic activity is determined by gating on target cell population (R1 + R2) fig 1. Creating a dot plot gated from fig 1, a plot of apoptosis vs. necrosis is derived fig 2. Apoptosis in induced early in the cytolytic process. By incorporating apoptosis, the total cytotoxicity is the most sensitive assay available fig 3.

Total Cytotoxicity Assay Kits Available:

PRODUCT NUMBER
TEST SIZE
Tcy125
125
Tcy250
250
Tcy500
500

Manual

Cell Calculation Spreadsheet

Referenceses: 


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

2. Slezak, S. and Horan, P.K., 1989. Cell-mediated cytotoxicity: A highly sensitive and informative flow cytometric assay. J. Immunol Methods 117: 205-214. 

3. 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 

4. Hatam, L,. Schuval, S., Bonagura, V.R., 1994. Flow cytometric analysis of natural killer cell function as a clinical assay. Cytometry 16:59. 

5. 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. 

6. Van Hooijdonk, 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. 

7. 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. 

8 Olin, M., Choi, K., Lee, J., and Molitor, T.W., 2004. γδT-lymphocytes cytolytic activity against Mycobacterium tuberculosis analyzed by flow cytometry. J. Immunol Methods, 2005, 297, 1-11. 

9. Bedner E., Smolewski P., Amstad P., Darzynkiewicz, Z., 2000. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 2000 Aug 25; 259(1): 308-13.

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AQ Molecular and Immunochemical