The overall goal of this procedure is to monitor NK cell functions using a flow cytometry-based assay. Natural killer cells are crucial for the outcome of various viral infections and malignant diseases. To better monitor NK cell functions, our lab has further developed a flow cytometry-based assay that can be used in both scientific research and clinical evaluations.
We optimized the protocol to permit the analysis of small NK cell sample sizes which is essential for its use in pediatrics and for immunodeficient patients. An additional advantage of this technique is the possibility to measure NK cell functions not only within the whole NK cell population, but also in different NK cell subsets. To isolate the NK cells, begin by mixing the appropriate volume of NK cell isolation cocktail solution with six to 10 milliliters of EDTA peripheral whole blood from a patient by several inversions.
Next, further homogenize the sample on a tube rotator for five minutes at room temperature. Then place the tube into a magnetic separator for 15 minutes under sterile conditions, taking care that the tube label faces toward the back of the magnet to facilitate the visibility of the separation line. At the end of the separation, carefully transfer the supernatant into a new 15 milliliter tube and bring the final volume up to 15 milliliters with complete medium.
Wash the cells by centrifugation and resuspend the pellet in one milliliter of fresh complete medium. Then count the cells and adjust the volume to a concentration of at least two times 10 to the fourth NK cells per 100 microliters in complete medium. To stimulate the isolated NK cells, first gently mix by pipetting at least five times and aliquot 100 microliters of the cells into the appropriate wells of a 96-well V-bottomed plate.
Switch off the light and add anti-CD107a antibody to each well of the NK cells. Then carefully mix an aliquot of K562 tumor cells at a two times 10 to the fourth cells per 100 microliter volume by pipetting at least five times and transfer 100 microliters of the tumor cells to each well planned for the stimulation. Precise pipetting and the preparation of a one-to-one effector target ratio are of key importance for the accurate assessment of NK cell function.
Next, add IL-2 and IL-15 to the stimulation wells and mix all of the wells by pipetting at least five times. Incubate the plate protected from light for three hours at 37 degrees Celsius and 5%carbon dioxide. After the first hour with the light switched off, add freshly prepared protein transport inhibitor solution to each well with careful mixing and return the plate to the incubator.
At the end of incubation, carefully mix the co-cultures and transfer them into their corresponding flow cytometry tubes. Add one milliliter of wash buffer to each tube for centrifugation and resuspend the pellets in 99 microliters of PBS plus one microliter of fixable dead cell marker per sample. Mix the cells by vortexing, followed by a 15-minute incubation at room temperature in the dark.
At the end of the incubation, wash the cells in one milliliter of wash buffer and resuspend the pellets in 84 microliters of wash buffer plus 16 microliters of the appropriate cell surface antibody cocktail. Mix the samples by vortexing and incubate for 20 minutes in the dark at four degrees Celsius. Then wash the cells in one milliliter of wash buffer and resuspend the pellets in 100 microliters of a cold fixation solution.
Mix by vortexing, followed by 10 minutes at four degrees Celsius in the dark. Now wash the cells in one milliliter of wash buffer and resuspend the pellets in 90 microliters of permeabilization buffer. Stain with 10 microliters of the appropriate intracellular antibodies and mix by vortexing.
After 30 minutes in the dark at four degrees Celsius, wash the cells two times in one milliliter of fresh permeabilization buffer. Then resuspend the samples in 400 microliters of fresh permeabilization buffer. Mix well by vortexing and place the cells on ice until their analysis by flow cytometry.
As soon as possible, transfer all tubes into the flow cytometer and start the data acquisition. Here, gating strategies for analyzing the de-granulation and cytokine and chemokine production of the whole NK cell population as well as three different NK cell subsets are illustrated. Unstimulated NK cells from healthy donors produced neither interferon gamma nor MIP-1 beta and do not express CD107a on their surface.
In contrast, NK cell stimulated with tumor cells and cytokines produced significant amounts of intracellular interferon gamma and MIP-1 beta with over 20%of the cells demonstrating de-granulation upon tumor cell interaction as indicated by their CD107a cell surface expression. Once mastered, this technique can be completed in eight hours. While attempting this procedure, it is important to be precise in the plating of the NK cells and K562 tumor cells.
To maintain the selected effector target ratio, the counting should be as accurate as possible. This flexible procedure can be easily modified. For example, NK cells within the PBMC population can be analyzed or different antibodies can be used to check for other markers of interest.
After watching this video, you should have a good understanding of how to purify, stimulate and label NK cells to monitor their function using a flow cytometry-based assay. Don't forget that working with human and target cells is potentially hazardous and that safety precautions such as wearing the appropriate personal protection equipment should always be taken while performing this procedure.
