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 facilitated by non-specifically lysing infected targets through the use of NK receptors, or the FcγII (CD16) receptor, recognizing IgG bound to specific antigens on the target cell surface. 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 51Cr from lysed target cells. Unfortunately, these techniques have several drawbacks. Traditional enzyme-release assays are often skewed by the large number of necrotic effector cells. Problems associated with 51Cr release methods include high spontaneous leakage resulting in high backgrounds, high cost, short half-life, and the health risks due to exposure to radioactive material. Beyond these limitations, these assays frequently underestimate the true level of cytotoxicity, as they are unable to detect early-stage apoptotic cells.
Flow cytometric assays have been developed to overcome some of the difficulties associated with older assays like lactate dehydrogenase and 51Cr release assays. Once such early version involved the detection of NK cyto- toxicity activity by staining target cells with the green fluorescent dye, F-18, in combination with the DNA intercalating dye, propidium iodide. 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. However, despite correlations of greater than 95% when compared with the 51Cr release assay, the PKH-26 method is 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.
More recently, the problems with older flow cytometric assays were overcome with the use of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), a green fluorogenic reagent that diffuses into target cells and covalently binds to primary amino groups on intracellular molecules. Intracellular esterases quickly cleave the acetate groups from the dye, thus converting it to its green fluorescent form. Any unbound reagent diffuses back out of the cell. Building upon these techniques, ImmunoChemistry Technologies has developed the Total Cytotoxicity & Apoptosis Assay, a flow cytometric assay combining the green fluorescing cellular stain, (CFSE), with a red fluorescing live/dead stain, 7-aminoactinomycin D (7-AAD), and ICT’s SR-FLICA® apoptosis detection reagent to concurrently quantify caspase-positive cells. The assay can be used to determine total cytotoxicity in the form of apoptosis and necrosis. It will quantify 4 populations of cells: live; early apoptotic; late apoptotic; and necrotic cells within a single sample tube.
While other methods often underestimate the true level of cytotoxicity, ICT’s Total Cytotoxicity & Apoptosis Assay is the best method to accurately quantify cell death because it can detect cells in early apoptosis. This often reveals a significant percentage of cells that are 7-AAD negative (indicating that they are alive and do not have compromised membranes), but are SR-FLICA® positive (meaning that they are becoming apoptotic and dying and have active caspase enzymes).
In the assay, CFSE is first used to label the target cell population green (Figure 4). The unstained effector cells are then added and incubated with the target cells (referred to as the Effector:Target, or ‘E:T’ mixture, Figure 5). As all the target cells are initially labeled with green fluorescing CFSE, and the effector cells are not, these two populations can be easily distinguished (Figure 8). Apoptotic target cells can then be identified by labeling with the second reagent, SR-FLICA (Figure 6). SR-FLICA® is an orange/red fluorescent poly caspase inhibitor, SR-FLICA®, which binds to active caspase enzymes up-regulated for apoptosis. Upon completion of the E:T incubation (which includes exposure to the apoptosis detection reagent), the last reagent, 7-AAD, is added to stain all dead cells red by binding to the DNA of membrane-compromised cells (Figure 7). With proper compensation and gating of the flow cytometer using the designated instrument controls (Figures 3 and 8-13) researchers can distinguish between target and effector cells, and living, necrotic, and apoptotic cells, and assess the level of cytotoxicity in their samples (Figures 14-15).
- Prepare samples and controls
- Dilute Assay Buffer 1:10 with diH2O and filter sterilize.
- Reconstitute CFSE with 200 µL DMSO and dilute 1:250 with 1X Assay Buffer.
- Add 200 µL CFSE to target cells to stain them green and incubate 15 minutes at room temperature.
- Wash target cells and adjust cell concentration.
- Add effector cells to target cells and incubate 4-6 hours.
- Reconstitute SR-FLICA with 100 µL DMSO and dilute 1:12.6.
- Add 10 µL SR-FLICA to label apoptotic cells orange-red and incubate 45 minutes at 37°C.
- Reconstitute 7-AAD with 260 µL DMSO and dilute 1:10 with 1X Assay Buffer.
- Add 20 µL 7-AAD to label necrotic cells red and incubate 10 minutes on ice.
- Run controls and analyze with a flow cytometer. Identify target cells by analyzing FL-1 (green) vs. FL-3 (red). Create a plot of FL-2 (orange-red) vs. FL-3 (red). CFSE excites at 492 nm and emits at 520-540 nm; 7-AAD excites at 546 nm and emits at 647 nm; SR-FLICA excites at 565 nm and emits at 600 nm.