Sep 01, 2022
Version 1
Assessing IL-15 bioavailability ("the bioassay") V.1
- Philippa R Kennedy1,
- Joshua T Walker1,
- Todd Lenvik1
- 1University of Minnesota
External link: https://doi.org/10.3389/fimmu.2023.1060905
Protocol Citation: Philippa R Kennedy, Joshua T Walker, Todd Lenvik 2022. Assessing IL-15 bioavailability ("the bioassay"). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmxqw9l3p/v1
Manuscript citation:
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:
Materials
MATERIALS
96 well black assay plate with clear bottomCorningCatalog #3603
ResazurinR&D SystemsCatalog #AR002
NK-92ATCCCatalog #CRL-247
Protocol materials
NK-92ATCCCatalog #CRL-247
Materials, Step 1
2-Mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M7522
Step 1.1
Horse Serum, heat inactivated, New Zealand originThermo FisherCatalog #26050088
Step 1.1
Fetal Bovine Serum, qualified, United StatesThermo FisherCatalog #26140079
Step 1.1
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
Step 1.1
Proleukin (recombinant IL-2)Catalog #National Drug Code 65483-116-07
Step 1.1
96 well black assay plate with clear bottomCorningCatalog #3603
Materials
ResazurinR&D SystemsCatalog #AR002
Materials, Step 6
NK-92 culture
NK-92 culture
Defrost NK-92 (malignant non-Hodgkin's lymphoma; see Cell Line Information) and culture for at least one week prior to initiation of the assay.
NK-92ATCCCatalog #CRL-247
NK-92 media
- Alpha Minimum Essential Medium plus ribonucleosides and deoxyribonucleosides
(Gibco Cat. No. 12571)
- 0.1 mM 2-mercaptoethanol (Sigma Aldrich, Cat. No. M7522)
- 12.5% horse serum (Fisher Scientific Cat. No. 26050088)
- 12.5% fetal bovine serum (FBS; Gibco Cat. No. 26140079)
- 100 U/mL penicillin streptomycin (Gibco Cat. No. 15140122)
For normal culture, supplement with 500 U/mL recombinant human IL-2 (Prometheus, Cat. No. NDC 65483-116-07).
2-Mercaptoethanol Sigma AldrichCatalog #M7522
Horse Serum, heat inactivated, New Zealand originThermo FisherCatalog #26050088
Fetal Bovine Serum, qualified, United StatesThermo FisherCatalog #26140079
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
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.
Software
Prism
NAME
GraphPad
DEVELOPER