The overall goal of this procedure is to assess the cytotoxic activity of human NK cells. This method can be used to monitor patients with either functional or classic NK cell deficiency. The main advantages of this flow cytometry based technique are that it's sensitive, reproducible, and faster than standard NK cell function assessment methods such as the radioactive chromium release assay.
This method can be used to test novel NK cell targeted therapies as well as previously unachievable in depth multi-parameter flow cytometric analysis of NK cell biology. Begin by using density gradient separation to isolate the peripheral blood mononuclear cells, or PBMC's from whole blood samples according to standard protocols. Using PBMC's instead of purified NK cells is greatly beneficial when dealing with clinical specimens.
Dilute the isolated cells to a five times 10 to the sixth cells per milliliter concentration in complete medium and immediately collect K562 target cells by centrifugation. Re-suspend five times 10 to the fifth 562 cells in 500 microliters of fresh medium supplemented with one percent FBS. Next label the target cells with 500 microliters of CFSE working solution and gently mix the cell solution by pipetting three to five times.
After 10 minutes at 37 degrees Celsius protected from light, quench the labeling reaction with 10 milliliters of complete medium for 10 minutes in the dark at room temperature. At the end of the quenching incubation collect the cells by centrifugation and re-suspend the labeled target cell palette in one milliliter of complete medium for counting. Then dilute the cells to one times 10 to the fifth cells per milliliter of fresh complete medium concentration and place the target cells in a humidified 37 degree Celsius and five percent carbon dioxide incubator.
For a successful assay, reduce the target cell stress to a minimum and test the effector cell as soon as possible after collection. Within 30 minutes of the target cell labeling, label a 96 well U bottom tissue culture treated plate according to the table. Next add 100 microliters of complete medium to the 25 to one, 12.5 to one, and 6.25 to one effector to target cell ratio wells, the target cells only wells, and the effector cells only wells.
Add 100 microliters of effector cells to the cytotoxcity control wells and the 50 to one, and 25 to one effector cell to target cell ratio wells. Then starting with the 25 to one effector cell to target cell ratio well, transfer 100 microliters of cells to the well with the next lowest cell ratio concentration for the next two wells, skipping the target cell only wells, mixing well between each cell transfer and adding the last 100 microliters of cells the the effector cells only wells. Add 100 microliters of 2X 0.2 percent Tween working solution to the Tween only K562 death positive control wells.
Add 30 microliters of vial two working solution to the NK cell cytotoxicity positive control wells. Then add one times 10 to the fifth labeled K562 target cells per milliliter in 100 microliters of complete medium to all of the wells except the effector cells only wells. When all the cells have been plated mix all the wells with the multi channel pipette.
Settle the cells with a brief centrifugation and place the plate in the cell culture incubator for four hours. At the end of the incubation place the plate on ice or on a cold platform, and add 50 microliters of freshly prepared dead cell stain to each well. Then gently mix all of the wells and analyze the cells by flow cytometry.
Flow cytometric analysis of CSFE and dead cell stain after four hours of cold culture as just demonstrated reveals that the cells in the target cells only wells experience 15%cell death or less. No CSFE signal is detected in the effector cells only wells if less than five percent cell death observed in these wells. While the cell death in the Tween mediated target cell killing wells is typically over 85%In patients with a defect in NK cell function a reduced killing activity is observed at most or all of the tested ratios compared to healthy individuals demonstrating that the NK cells from these patients exhibit a differential reduction in cytotoxicity that correlates with a decreasing effector target cell ratio.
Once mastered this technique can be completed in six and a half hours, including the two hours of preparation and plating, four hours of incubation, and 30 minutes of acquisition. While attempting this procedure it's important to remember to prepare the target and effector cells in parallel to minimize the lag time between the cell preparation and the start of the co-incubation. After watching this video you should have a good understanding of how to measure the cytotoxic activity of human NK cells against fluorescently labeled target cells by flow cytometry.
