Assessing IL-15 bioavailability ("the bioassay")
Philippa R Kennedy, Joshua T Walker, Todd Lenvik
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:
https://linkinghub.elsevier.com/retrieve/pii/0022175994903964
Steps
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-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).
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
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
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
After 24 h, add 10 µL resazurin to each well, mix and incubate for 1 h at 37℃ and 5% CO2.
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
| Value | Label |
|---|---|
| Prism | NAME |
| GraphPad | DEVELOPER |
| https://www.graphpad.com/scientific-software/prism/ | LINK |
| 8.0 | VERSION |
