APPLICATION NOTE

Quantification of functional IL-12 usingiLite® IL-12 Assay Ready Cells
For research and professional use only. Not for use in diagnostic procedures.
This application note contains a suggested protocol and performance data.
Each individual laboratory must set up their own method and perform relevant validations.

Background
Interleukin-12 (IL-12) is a 70 kDa cytokine mainly produced by macrophages, neutrophils and dendritic
cells in response to stimulation by inflammatory antigens. Stimulation of T-cells, involvement in T-celldifferentiation of Th1 cells and stimulation of IFN-gamma and TNF-alpha production are key functionsof IL-12. IL-12 is composed of two subunits, p35 and p40, covalently linked by a single disulfide bond.
The p40 subunit, which binds to the receptor chain IL-12Rβ1, is shared with another heterodimericcytokine, IL-23. However, the two cytokines expert distinct non-redundant biological functions (1).
Therapeutic agents targeting both IL-12 and IL-23 cytokines are currently used to treat psoriasis andpsoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders (2).

Principle of the assay

The iLite® IL-12 Assay Ready Cells are engineered cells, optimized to express Firefly luciferase (FL)under the control of an IL-12 responsive promoter. Binding of IL-12 to the IL-12 receptor (IL12R1 andIL12R2) results in activation of the IL-12 regulated Firefly luciferase reporter gene construct.
iLite® IL-12 Assay Ready Cells also contain the Renilla Luciferase (RL) reporter gene, under the control
of a constitutive promoter. The constitutive expression of RL allows normalization of IL-12 induced FL
activity, and renders assay results independent of variations in cell number or serum matrix effects. Theluciferase signal can be measured in a luminometer following addition and incubation of luciferase
substrate. The Firefly luciferase signal is proportional to the concentration of functional IL-12 in a sample(Fig.1).

Specimen collection
The iLite® IL-12 Assay Ready Cells can be used for measuring concentration of IL-12 in test samples
including human serum.

Material and equipment needed
Material and equipment

Suggested supplier

ReferenceiLite® IL-12 Assay Ready Cells
Diluent (RPMI containing 9% heat inactivated FBS +1% Penicillin-Streptomycin)

Svar Life Science
Gibco

IL-12 or analogues
Firefly/Renilla luciferase substrate

R&D Systems
PromegaPlate; White walled micro well plate suitable forluminescence
Microplate Luminometer with appropriate reading
software – no filter on luminometer

PerkinElmer

BM401261870-044 (RPMI)
26140-079 (FBS)
15140-122 (PenicillinStreptomycin)
219-IL
E2920, Dual-Glo
Luciferase Assay System
6005680

Incubator, 37 C with 5% CO2

Contact Svar Life
Science for list ofrecommended suppliers
NANANA

Svar Life Science AB
Lundavägen 151P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0477, 1.0

APPLICATION NOTE
Water bath, 37 C
Single-channel and multi-channel pipettes with
polypropylene disposable tips
Polypropylene tubes or plate for dilution
Single-use polypropylene reservoir
Plate shakerTimer

NA
NA

NA
NA

NA
NA
NA
NA

NA
NA
NA
NA

ProtocolPreparation of calibrators (IL-12)
Recombinant IL-12 from R&D Systems has successfully been used to stimulate the iLite® IL-12 Assay
Ready Cells. The below table shows the dilutions of IL-12, used for QC release of the iLite® IL-12 Assay
Ready Cells.
Figure 1. Example of IL-12 calibration curve.

Relative light units

200000Calibrator

150000

A100000

BC
D

50000
00.001

0.01

0.1

110

E
F

100

G
H

IL-12 (final conc.ng/mL)

IL-12
Suggestedcalibrator solution
conc. (ng/ml)
56
14
3.5
0.87
0.22
0.054
0.014
0

Table 1. Suggested calibrator solution
concentrations for IL-12.

Assay preparation and incubation
1. Design a plate layout. It is recommended to perform the test at least in duplicates.
2. Dilute calibrators, controls and samples to fall within the expected in assay values of0-28 ng/mL.
3. Add 40 µL calibrators, controls and samples in duplicate to assigned wells (final concentration
will be half of the solution concentration).
4. Thaw a vial of iLite® IL-12 Assay Ready Cells in a water bath at 37C with gentle agitation. The
cell suspension is mixed very carefully ten times with a pipette to ensure a homogeneous
distribution of cells.
5. Dilute 250 µL cell suspension with 5.75 mL Diluent.
6. Add 40 µL diluted cells to each well.
7. Place the lid on the plate, mix and incubate for 5 hours at 37 °C with 5% CO2.
Adding substrate solutions
8. Equilibrate the plate and the substrate solution to room temperature.
9. Prepare the Firefly luciferase substrate according to the supplier’s instructions and add
80 µL per well. Mix and protect the plate from light. After 10 minutes incubation at room
temperature read in a luminometer.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0477, 1.0

APPLICATION NOTE
10. If appropriate, prepare the Renilla luciferase substrate according to the supplier’s instructions
and add 80 µL per well. Mix and protect the plate from light. After 10 minutes incubation at room
temperature read in a luminometer.

Precautions






This application note is intended for professional laboratory research use only. The data andresults originating from following the Application Note should not be used either in diagnostic
procedures or in human therapeutic applications.
Use and handle the material and instruments referenced according to the
supplier’s/manufacturer’s instructions or product specifications accompanying the individualmaterial and instruments.
Dispose of all sample specimens, infected or potentially infected material in accordance withgood microbiological practice. All such materials should be handled and disposed as thoughpotentially infectious.
Residues of chemicals and preparations are generally considered as biohazardous waste andshould be inactivated prior to disposal by autoclaving or using bleach. All such materials should
be disposed of in accordance with established safety procedures.

Proprietary Information
In accepting delivery of iLite® Assay Ready Cells the recipient agrees not to sub-culture these cells,attempt to sub-culture them or to give them to a third-party recipient, and only to use them directly inassays. iLite® cell-based products are covered by patents which are the property of Svar Life Science
AB and any attempt to reproduce the delivered iLite® Assay Ready Cells is an infringement of thesepatents.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0477, 1.0

APPLICATION NOTEQUICK GUIDE
Quantification of functional IL-12 using iLite® IL-12 Assay Ready Cells

1Sample dilution• Equilibrate reagents and samples to room temperature – do not thaw cells and substrate
reagents at this stage• Dilute calibrators, controls and samples.
• Add 40 µL of calibrators, controls and diluted samples to pre-assigned wells.
• Thaw the cell vial in a 37C water bath. Mix the cell suspension with a pipette in order to ensure ahomogeneous cell solution. Dilute the cells.
• Add 40 µL diluted cells to each well.

• Incubate at 37 °C with 5% CO2 for 5 hours.

2Incubation
5h3
Read plate• Equilibrate the plate to room temperature• Prepare the Firefly luciferase substrate according to the supplier's instructions and add 80 µL perwell. Mix. Protect the plate from light. After 10 min incubation read in a luminometer.
• If appropriate, prepare the Renilla luciferase substrate according to the supplier's instructions andadd 80 µL per well. Mix. Protect the plate from light. After 10 min incubation read in a luminometer.

Troubleshooting and FAQ
Please consult the Svar Life Science website www.svarlifescience.com

References
1. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. (April 1993).
Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.
Science 260: 547–5499 (1993).
2. Teng MW, Bowman EP, McElwee JJ, Smyth MJ, Casanova JL, Cooper AM, Cua DJ. IL-12
and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory
diseases. Nature Medicine 21: 719–729 (2015).

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0477, 1.0