A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

This method helps to compare the cytotoxic activity of different cell types using a simple, inexpensive method that does not require specialized equipment. The main advantage of this technique is that it doesn't require the use of hazardous materials, it is inexpensive, and does not require the use of specialized equipment. This method can be applied to measure cytotoxic activity of other cell types such as CD8 plus cytolytic cells or even to assess spontaneous death of cells without incubating, without cytolytic effector cells.

Individuals new to this technique will likely struggle with the optimization of target to effector cell ratio. My advice is to do a thorough search of the literature to determine the ratios that you would test when optimizing the protocol. Although this method does not require specialized skills, other than pipetting accurately, the isolation of mononuclear cells and the setup of the assay plate are steps that can be easier understood and mimicked if they are observed visually.

Demonstrating the procedure will be Chelsea Jekelley, a technician from my laboratory. To begin this procedure, add 13.5 milliliters of RPMI in 1.5 milliliters of FBS to a Petri dish containing a 100 micrometer filter. Place one placenta on the filter and use the flat side of a syringe plunger to push it through the filter and into the Petri dish.

Next, set out three 15-milliliter conical tubes for each tissue. Add three milliliters of density gradient medium to each tube, and slowly overlay five milliliters of homogenized placenta into each tube. Centrifuge at 300 times G and at room temperature for 25 minutes with no break.

Then, use a transfer pipette to collect the thin white buffy layer. Add 10 milliliters of RPMI to the combined buffy layers. Centrifuge at 300 times G and at four degrees Celsius for 10 minutes and discard the supernatant.

First, re-suspend the cell pellet in 50 microliters of ice-cold PBS. Add biotin-labeled CD3 antibody according to the manufacturer's protocol and mix well with a pipette. Place the tube into a tube rotator and incubate at four degrees Celsius for 20 minutes.

Next, add one milliliter of RPMI. Centrifuge at 400 times G and at four degrees Celsius for 10 minutes. Discard the supernatant and re-suspend the pellet in one milliliter of RPMI.

Combine the cell suspension with 150 microliters of magnetic beads in a 1.5 milliliter microcentrifuge tube. Transfer the tube to a tube rotator and rotate while incubating at four degrees Celsius for 30 minutes. During the incubation, retrieve the release buffer and let it reach room temperature in a bio safety cabinet.

After the incubation is complete, place the tube in the magnet for one minute. Collect the supernatant and save the CD3 negative cell population in a 15 milliliter tube on ice. Remove the tube from the magnet, add one milliliter of RPMI and use a pipette to mix the cells and beads five times.

Then, place the tube back into the magnet for one minute. Collect the supernatant and save the CD3 negative population in a 15 milliliter tube on ice. Centrifuge the CD3 negative population of cells at 400 times G and at four degrees Celsius for 10 minutes.

Discard the supernatant and re-suspend the cell pellet in 50 microliters of ice-cold PBS. After this, add biotin-labeled CD161A antibody to the CD3 negative cells, according to the manufacturer's instructions and mix well. Place the tube in a tube rotator and incubate at four degrees Celsius for 20 minutes.

Next, add one milliliter of RPMI and centrifuge at 400 times G and at four degrees Celsius for 10 minutes. Discard the supernatant and re-suspend the pellet in one milliliter of RPMI and combine it with magnetic beads in a 1.5 milliliter microcentrifuge tube. Place the tube in tube rotator and rotate while incubating at four degrees Celsius for 30 minutes.

Then, place the tube in the magnet for one minute. Collect the supernatant and discard CD3 negative CD161A negative cells. Remove the tube from the magnet, add one milliliter of RPMI and mix well.

Place the tube in the magnet for one minute, making sure to collect the supernatant and discard the CD3 negative CD161A negative cells. After this, remove the tube from the magnet and add one milliliter of room temperature release buffer. Place the tube on a tube rotator and rotate while incubating for 15 minutes at room temperature.

Next, place the tube in the magnet for one minute. Collect the supernatant in a new 15-milliliter conical tube on ice. Remove the tube from the magnet, add one milliliter of room temperature RPMI and mix well.

Place the tube back under the magnet for one minute, making sure to collect the supernatant in the same 15-milliliter conical tube. Mix well and take a 20 microliter sample to count the cells. Centrifuge the cells at 400 times G and at four degrees Celsius for 10 minutes.

Remove the supernatant and re-suspend the cells in RPMI. Seed the cells into a six well plate at a concentration of 300, 000 cells per well in 2.5 milliliters of NK cell activation media. Incubate at 37 degrees Celsius with 5%carbon dioxide in a humidified incubator for 48 hours.

In a hood, use a serological pipette to transfer YAC1 cells in media to a 50 milliliter tube on ice and mix well. Take a 20 microliter aliquot to count the cells. Spin the cells at 300 times G and at four degrees Celsius for 10 minutes.

Using a serological pipette, collect the supernatant from the previously-prepared NK cell six well plate in 15 milliliter conical tube. Then, add trypsin EDTA to each well. Tap the plate and place it in an incubator at 37 degrees Celsius.

After the cells have incubated with trypsin EDTA for approximately five minutes, use a sterile plate scraper to scrape the plate. Then add one milliliter of NK cell media to each well. Using a serological pipette, collect the cells and media and transfer them into a 15-milliliter centrifuge tube.

Take a 20 microliter aliquot to count the cells. Centrifuge the NK cells at 400 times G and at four degrees Celsius for 10 minutes. First, obtain a round bottom, culture treated 96 well plate and set it up as outlined in table one of the text protocol.

Centrifuge the assay plate at 250 times G for four minutes to be certain that the effector and target cells are in contact. Next, incubate the plate in a humidified chamber at 37 degrees Celsius with 5%carbon dioxide for five hours. 45 minutes prior to harvesting the supernatants, add 10 microliters of 10X lysis solution to the target cell maximum LDH release wells and return the plate to the humidified chamber.

When the incubation is complete, centrifuge the plate at 250 times G for four minutes, then use a multi-channel pipetter to transfer 50 microliter aliquots from every well to a fresh 96-well, flat bottom assay plate. Add 50 microliters of assay reagent to each well of the assay plate. Cover the plate with foil to protect it from light and incubate at room temperature for 30 minutes.

After this, add 50 microliters of Stop Solution to each well. Make sure to read the plate within one hour of adding the Stop Solution and record the absorbance at 490 nanometers. Placental NK cells obtained from NP and RUPP rats are incubated for five hours with target cells in their respective medias at a ratio of 50:1.

The raw absorbance data recorded at 490 nanometers is shown here. The average absorbance of the cultural medium background and the volume correction control wells are calculated, and these averages are subtracted from the appropriate wells indicated in the manufacturer's protocol. The corrected values are then used to obtain the cytotoxicity using the protocol provided by the manufacturer.

Although this method does not require specialized skills, other than pipetting accurately, the isolation of mononuclear cells and the setup of the assay plate are steps that can be easier understood and mimicked if they are observed visually. After isolation of the NK cells, a number of other procedures could be performed, such as co-culture experiments to determine the role of placental NK cells in attributing to the trophoblast migration during pregnancy. We also collect the media from the expanded NK cells to assess cytokinase secreted from the differentially stimulated cells.

The purity of the isolated cells could also be assessed via flow cytometry.