TITLE: Assessing IL-15 bioavailability (&#34;the bioassay&#34;)
AUTHORS: Philippa R Kennedy, Joshua T Walker, Todd Lenvik

[DESCRIPTION]
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:
https://linkinghub.elsevier.com/retrieve/pii/0022175994903964

http://doi.wiley.com/10.1046/j.1440-1711.1998.00733.x

[STEPS]
SECTION: NK-92 culture
1. 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.
 

SECTION: NK-92 culture
1.1. 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).
 

 

 

 

 

SECTION: NK-92 culture
1.2. 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
SECTION: NK-92 culture
1.3. 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

SECTION: Day - 1
2. Deprive NK-92 of IL-2 at least 16 h prior to plating cells.
SECTION: Day 0
3. 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).
SECTION: Day 0
4. 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.
SECTION: Day 0
5. Add 20 μL of diluted drug to each well containing NK-92. Include a no drug condition as a negative control.
SECTION: Day - 1
2.1. Count NK cells (at least 4.8 million will be required for a full plate).
SECTION: Day - 1
2.2. Spin NK-92 at 1600 RPM for 5 min and remove the supernatant.
SECTION: Day - 1
2.3. Resuspend cells in media without IL-2.
SECTION: Day - 1
2.4. Spin NK-92 at 1600 RPM for 5 min and remove supernatant. 
SECTION: Day - 1
2.5. Resuspend cells at 7.5 x 105cells/mL in media without IL-2 and return to the incubator overnight.
SECTION: Day 2
6. After 24 h, add 10 µL resazurin to each well, mix and incubate for 1 h at 37℃ and 5% CO2.
 

SECTION: Day 2
8. 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).
SECTION: Day 2
9. 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. 
 

SECTION: Day 2
7. 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).