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

All protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. The care and handling of the animals were in accord with the National Institutes of Health guidelines for ethical animal treatment.

1. Lymphocyte Cell Isolation from Placentas

1. Remove one placenta from the rat uterus (gestation day 19) and place in 10 mL of ice-cold PBS⁸.
2. Place one placenta on a 100 µm filter and sit in a Petri dish containing 13.5 mL of RPMI and 1.5 mL of  FBS (total volume is 15 mL). Use the flat side of a syringe plunger to push the placenta through the filter into the Petri dish.
3. Prepare three 15 mL conical tubes for each tissue. Add 3 mL of density gradient medium (see Table of Materials) to each tube, then carefully overlay 5 mL of homogenized placenta into each tube.
4. Centrifuge for 25 min at 300 x g at room temperature (RT) with no brake. Collect the thin white buffy layer with a transfer pipette.
   NOTE: After centrifugation, 3 layers are visible, the red RPMI at the top, the white buffy layer in the middle, and the clear density gradient medium layer at the bottom. Use a transfer pipette to pull up the white buffy layer from the tube. Combine buffy layer from all tubes of the same placenta into 1 tube.
5. Add 10 mL of RPMI to combined buffy layers. Centrifuge for 10 min, 300 x g, at 4 °C and discard supernatant.

2. Isolation of Natural Killer Cells

1. Resuspend cell pellet in 50 μL of ice-cold PBS
2. Add biotin-labeled CD3 antibody to pelleted cells according to the manufacturer’s protocol and mix well with a pipette. Place the tube in a tube rotator and incubate 20 min at 4 °C.
3. Add 1 mL of RPMI, centrifuge for 10 min at 400 x g and 4 °C, and discard supernatant.
4. Resuspend pellet in 1 mL of RPMI and combine with 150 μL of magnetic beads in a 1.5 mL microcentrifuge tube. Place the microcentrifuge tube in a tube rotator and rotate while incubating for 30 min at 4 °C.
   NOTE: Pull out release buffer at this time and allow to reach RT in the biosafety cabinet.
5. Place the tubes in the magnet for 1 min. Collect supernatant and save CD3- cell population in a 15 mL tube on ice.
   NOTE: This is the CD3^– population of cells.
6. Remove the tube from the magnet and add 1 mL of RPMI. Mix cells and beads 5 times with a pipette.
7. Repeat step 2.5.
8. Centrifuge the CD3^– population of cells for 10 min at 400 x g and 4 °C and discard supernatant. Resuspend the cell pellet in 50 μL of ice-cold PBS
9. Add biotin-labeled CD161a antibody to CD3^– cells according to the manufacturer’s protocol and mix well. Place tube in a tube rotator and incubate for 20 min at 4 °C.
10. Add 1 mL of RPMI, centrifuge for 10 min at 400 x g and 4 °C, and discard supernatant. Resuspend pellet in 1 mL of RPMI and combine with 150 μL of magnetic beads in a 1.5 mL microcentrifuge tube.
11. Place microcentrifuge tube in a tube rotator and rotate while incubating for 30 min at 4 °C.
12. Place the tubes in a magnet for 1 min. Collect supernatant and discard CD3^–/CD161a^– cells.
13. Remove tube from magnet and add 1 mL of RPMI. Mix cells and beads 5 times with a pipette.
14. Repeat step 2.12.
15. Remove microcentrifuge tubes from the magnet and add 1 mL of RT Release buffer. Place tube on tube rotator and rotate while incubating for 15 min at RT.
16. Place tubes in magnet for 1 min. Collect supernatant in a new 15 mL conical tube on ice.
    NOTE: This is the population of NK cells.
17. Remove tube from magnet and add 1 mL of RT RPMI. Mix cells and beads 5 times with pipette.
18. Repeat step 2.16, placing supernatant in the same tube. Mix well and take a 20 μL sample to count cells
    NOTE: Keep tube on ice.
19. Centrifuge CD3^–/CD161a⁺ cells for 10 min, 400 x g at 4 °C, then remove supernatant. Resuspend the cells in RPMI (10% FBS, 1% Pen/Strep, 2 ng/ mL IL-2) and seed at a concentration of 3 x 10⁵ cells/well in a 6-well plate in 2.5 mL of NK Cell Activation Media. Incubate cells for 48 h at 37 °C, 5% CO₂ in a humidified incubator.

3. Cytotoxicity Assay: Retrieving NK Cells or YAC1 from Culture or Passing Cells

NOTE: All steps must be conducted under the hood. All cell tubes must be kept on ice at all times.

1. Use a glass serological pipet to collect YAC1 cells and media from flask, place in a 50 mL tube on ice, and mix well. Take 20 µL to count cells.
2. Spin the YAC1 cells for 10 min at 300 x g and 4 °C. Count cells while these are spinning.
3. Add trypsin/EDTA to each well in a NK cell 6-well plate. Tap the plate and place in incubator.
4. After cells have incubated with trypsin/EDTA for ~5 min at 37 °C, scrape the plate/flask with a sterile plate scraper. Add 1 mL of NK Cell Media to each well.
5. Collect cells and media with serological pipet and collect in a 15 mL centrifuge tube. Take 20 µL to count cells.
6. Spin the NK cells for 10 min, 400 x g at 4 °C. Count the sample of cells from step 3.5 during this centrifugation.
   NOTE: View the culture plates under a microscope before discarding them to make sure there are no more cells adhered to the bottom of the wells. Since the experiment uses NK cells and YAC1 cells, be sure to count each one separately.
7. Count cells and resuspend at the concentrations determined in optimization trials to test for the appropriate number of target:effector ratio. This will be achieved by re-suspending the pellet in their corresponding media to make the following cell concentrations: YAC1 at 4 x 10⁵ cells/mL and NK cells at 2 x 10⁷ cells/mL.

4. Cytotoxicity Assay: Assay Protocol

1. Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows the experimental controls and 3 sets of NK experimental columns. This can be expanded to a total of 10 NK experimental columns in a 96-well plate.
2. Centrifuge the assay plate at 250 x g for 4 min to be certain that the effector and target cells are in contact. Incubate the 96-well plate for 5 hours in a humidified chamber incubator at 37 °C, 5% CO₂ to achieve ample contact between target and effector cells and target cell lysis by effector cells.
   NOTE: The protocol can be paused here.
3. 45 min prior to harvesting supernatants, add 10 µL of 10x Lysis Solution to the Target Cell Maximum LDH Release wells (Wells 1E, 1F, 1G, and 1H) and place the plate back in the humidified chamber.
4. After the incubation time is completed, centrifuge the plate at 250 x g for 4 min. Using a multichannel pipettor, transfer 50 µL aliquots from all wells to a fresh 96-well flat-bottom assay plate.
   NOTE: Do not touch the bottom of the wells so that cells are not transferred into the fresh assay plate. Do not use a cultured treated plate as the fresh assay plate.
5. Make Assay Reagent.
   1. After Assay Buffer has reached room temperature, add 12 mL of Assay Buffer to one Substrate Mix bottle. Invert and shake gently until completely dissolved. 1 bottle is enough for two 96-well plates.
      NOTE: Assay Buffer should be protected from light while being thawed. Mix immediately prior to use.
6. Add 50 µL of Assay Reagent to all wells in the assay plate from step 4.6. Cover the plate with foil to protect it from light and incubate for 30 min at room temperature.
   NOTE: Newly made Assay Reagent should be stored in a freezer. Reagent is good for approximately 8 weeks.
7. Add 50 µL of Stop Solution to each well. Read plate within 1 hour after adding Stop Solution and record the absorbance at 490 nm. Representative data is shown in Table 2.

5. Calculation of Results

1. Calculate the average absorbance value from the Culture Medium Background wells and subtract from all absorbance values for Experimental, Target Cell Spontaneous LDH Release and Effector Cell Spontaneous LDH Release wells.
2. Calculate the average absorbance values for the Volume Correction Control wells and subtract from the absorbance values acquired for the Target Cell Maximum LDH Release Control wells.
3. Use the corrected values from Steps 5.1 and 5.2 in the following formula to calculate percent cytotoxicity for each effector:target well.