An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

1. 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.

2. 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.
2. 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.
3. 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.

Table 1: Assay Plate Layout. TCS = Target cell Spontaneous LDH Release (50 μL target cells + 50 μL target cell medium); TCM = Target Cell Maximum LDH Release (50 μL target cells + 50μL target cell medium); VCC = Volume Correction Control (50 μL target cell medium + 50 μL NK Cell medium); CMB = Culture Medium Background Control (50 μL target cell medium + 50 μL NK Cell medium); ECSR = Effector Cell Spontaneous Release (50 μL NK cells + 50 μL NK cells medium); EW = Experimental wells (50 μL target cells + 50 μL NK cells).

Table 2: Raw absorbance data of an assay plate containing the experimental controls in columns 1 and 2 and experimental wells to measure the cytotoxicity of placental NK cells from NP and RUPP rats against YAC1 target cells.