We are going to demonstrate two protocols today that demonstrate how to study NK cell function. There are two key aspects of NK cell function that is it's able to eradicate tumor cells and it's able to migrate to tumor micro environment. These two protocols we'll demonstrate NK cell ability to eradicate tumor cells and also it's able to migrate to tumor micro environment.
So the two protocols that we are going to demonstrate today are straightforward, does not require use of radio activity and can be established in most labs that has the ability to do molecular biology and cell culture work. Suresh Bugide and Suresh Chava, who are two postdoctorate associates in my lab, will demonstrate these two protocols today. Two assess NK cell mediated cytotoxicity using LDH, first grow a human liver cancer cell line to 70-80%confluency in an 100 millimeter cell culture Petri dish at 37 degrees Celsius and 5%carbon dioxide.
On the day of the experiment, wash the culture with five milliliters of PBS before treating the cells with one milliliter of 0.25%trypsin EDTA until the cells have detached from the plate bottom. When a single cell suspension has been obtained add 10 milliliters of cell culture medium to the cells and transfer the cells to a 15 milliliter conical tube for centrifugation. Wash the cell pellet with five milliliters of PBS before re-suspending the cells at a one times 10 to the 5th cells per milliliter of fresh culture medium concentration.
Next, seed 100 microliters of the target human liver cancer cells per well in triplicate per treatment condition in a 96 well plate and add one times 10 to the 5th human NK cells in 100 microliters of medium to each target cell well. Then place the plate in the cell culture incubator for three hours. At the end of the incubation, pellet the cells by centrifugation and transfer 100 microliters of supernatant from each well into individual wells of a new 96 well plate.
Add 50 microliters of LDH substrate to each well with thorough mixing and incubate the plate for 20 minutes at room temperature protected from light. At the end of the incubation, arrest the reaction with 50 microliters of stop solution and immediately measure the absorbance on a plate reader at 490 and 680 nanometers. Then use the formula to calculate the percent NK cell cytotoxicity.
To assess NK cell mediated cytotoxicity using calcein AM after growing human liver cancer cell line cells to a 70-80%confluency as demonstrated re-suspend the cells in three milliliters of serum-free DMEM and label the cells with 1.5 microliters of 10 millimolar calcein AM solution for 30 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation and wash the cells two times with five milliliters of PBS per wash. Re-suspend the cells at a one times 10 to the 5th cells per milliliter of culture medium concentration and add 100 microliters of cells to each well of a 96 well plate in triplicate per treatment condition.
Next, at one times 10 to the 5th human NK cells in 100 microliters of culture medium to each well of target cells and place the plate in the cell culture incubator for four hours. After the end of the incubation, capture at least 10 different fields of images of the calcein AM positive cells for each replicate of each treatment condition on a fluorescence microscope at a 10 times magnification. Then calculate the percent cytotoxicity using the formula.
To measure NK cell migration in response to chemotactic stimuli collect human NK cells from a 70-80%confluent culture and re-suspend the cells at a 2.5 times 10 to the 6th cells per milliliter of serum-free NK cell culture medium concentration. Next, add 600 microliters of serum-free medium containing the NK cell chemo attractant of interest per well and place one 6.5 millimeter diameter culture insert with five micrometer pores into each well of medium. Then add 100 microliters of NK cells to the upper compartment of each insert and place the plate in the cell culture incubator for four hours.
At the end of the incubation transfer the entire volume of non-adherent migrated cells from the bottom of each well into individual five milliliter FACS tubes and add 50 microliters of a predetermined number of flow cytometry counting beads to each tube. Then using a flow cytometer evaluate each cell sample according to standard flow cytometric analysis protocols and calculate the absolute number of migrated NK cells using the formula. ATF4 knockdown significantly reduces NK cell-mediated cytotoxicity compared to NK cells expressing non-specific short hairpin RNA.
After staining with calcein AM the number of viable human liver cancer cells decreases after co-culture with human NK cells compared to human liver cancer cells cultured without NK cells. As expected, ATF4 knockdown via short hairpin RNA reduces NK cell-mediated killing of human liver cancer cells as evidenced by a greater number of calcein AM-positive cells in these cultures. In addition, a significantly higher amount of NK cell migration is observed in response to chemokine-containing medium compared to NK cells exposed to control medium without chemokine.
For NK cell cytotoxicity assess it is important to standardize our effort and target ratios and incubation time for each cancer cell line. Once standards are identified and validated in vitro assess are supplemented with in vivo mice experiments to further evaluate their role in tumor cell eradication.
