Kennedy PR, Vallera DA, Ettestad B, Hallstrom C, Kodal B, Todhunter DA, Bendzick L, Hinderlie P, Walker JT, Pulkrabek B, Pastan I, Kratzke RA, Fujioka N, Miller JS, Felices M, A tri-specific killer engager against mesothelin targets NK cells towards lung cancer. Frontiers in Immunology doi: 10.3389/fimmu.2023.1060905
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2020
Last Modified: September 01, 2022
Protocol Integer ID: 35984
Abstract
Assessing the capacity of IL-15 analogs (e.g. within TriKE molecules) to stimulate proliferation of NK-92 cells. NK-92 are deprived of cytokines overnight, then cultured with IL-15 analogs for two days. After this time, the expansion of viable cells is quantitatively assessed using a redox-sensitive dye that changes from blue/non-fluorescent in media to pink/fluorescent upon reduction in viable cells.
It should be noted that NK-92 lack CD16, so delivery of IL-15 to these cells should not be enhanced by the anti-CD16 component of the TriKE as it would be for CD16+ NK-92 or CD16+ pNK cells.
This protocol is adapted from the following assays:
Proleukin (recombinant IL-2)Catalog #National Drug Code 65483-116-07
NK-92 culture conditions:
Humidified incubator at 37.0°C, 5% CO2
Culture conditions: Replace medium every 2 ‐ 3 days
Passage interval: Passage 2 ‐ 3 times a week
Sub-cultivation ratio: 1:2 - 1:3
Seeding conditions: 2x105 - 3x105 cells/mL
NK-92 freezing protocol:
Freeze medium: 50% FBS; 40% NK92 media; 10% DMSO
Freeze no more than 5x106 cells/mL
Storage temperature: liquid nitrogen vapor phase
Day - 1
Day - 1
Deprive NK-92 of IL-2 at least 16 h prior to plating cells.
Count NK cells (at least 4.8 million will be required for a full plate).
Spin NK-92 at 1600 RPM for 5 min and remove the supernatant.
Resuspend cells in media without IL-2.
Spin NK-92 at 1600 RPM for 5 min and remove supernatant.
Resuspend cells at 7.5 x 105cells/mL in media without IL-2 and return to the incubator overnight.
Day 0
Day 0
Dilute all drugs in media (without IL-2) to 5x the highest concentration to be tested (e.g. 150 nM). This will be used to make a serial dilution of the drug (3x, 5x or 10x depending on range required).
Plate NK-92 in triplicate into a 96 well black assay plate at 5 x 104 cells/well in 80 μL of media without IL-2. Avoid using the wells at the edge of the plate at these undergo the greatest evaporation.
Add 20 μL of diluted drug to each well containing NK-92. Include a no drug condition as a negative control.
Day 2
Day 2
After 24 h, add 10 µL resazurin to each well, mix and incubate for 1 h at 37℃ and 5% CO2.
ResazurinR&D SystemsCatalog #AR002
Ensuring there are no air bubbles in the media, read the fluorescence at 530-570 nm, with a correction at 590-620 nm using a plate reader (Tecan).
Replace the plate in the incubator and wait 1 h before repeating step 7. Repeat this process to obtain four readings in total. Select the most stable readings for analysis (usually 3 h).
Analyze the adjusted readings using Graphpad Prism. Log transform the data and calculate a non-linear fit using "log(agonist) vs. normalized response -- Variable slope - Least squares fit" in order to obtain an EC 50.