IL-8 Luc assay protocol

This protocol describes how to maintain the cells, how to prepare test chemicals, and how
to measure the luciferase activity of a THP-1-derived IL-8 reporter cell line, THP-G8, for
the IL-8 Luc assay.

1

Contents
1. Materials ...................................................................................................................4
1-1

Cells ....................................................................................................................4

1-2

Reagents and equipment .....................................................................................4

1-3

Culture medium..............................................................................................6

1-3-1

A medium: for maintenance of THP-G8 cells .............................................6

1-3-2

B medium: for luciferase assay ...................................................................6

1-3-3

C medium: for thawing THP-G8 cells .........................................................6

1-4

Thawing of THP-G8 cells................................................................................7

1-5

Maintenance of THP-G8 cells..........................................................................7

2. Preparation of cells for assay ..................................................................................8
3. Preparation of chemicals and cell treatment with chemicals ..............................9
3-1

Dissolution by X-VIVOTM 15 .............................................................................9

3-2

When the chemical is soluble at 20 mg/mL in X-VIVOTM 15 .........................12

3-2-1

Arrangement of chemicals and vehicle (1st experiment) .............................123-2-2

Serial dilution (1st experiment) ....................................................................123-2-3Addition to the cells (1st experiment) ........................................................13

3-2-4

Final constituents of each well of the plate (1st experiment) .......................14

3-2-5

Arrangement of chemicals and solvent (2nd , 3rd and 4th experiment) ..........15

3-2-6

Serial dilution (2nd , 3rd and 4th experiment) .................................................15

3-2-7

Addition to the cells (2nd , 3rd and 4th experiment) .....................................16

3-2-8

Final constituents of each well of the plate (2nd , 3rd and 4th experiment) ....17

3-3

When the chemical is not soluble at 20 mg/mL in X-VIVOTM 15 ...................18

3-3-1

Arrangement of chemicals and vehicle (1st experiment) .............................18

3-3-2

Serial dilution (1st experiment) ....................................................................183-3-3

Addition to the cells (1st experiment) ..........................................................192

3-3-4

Final constituents of each well of the plate (1st experiment) .......................20

3-3-5

Arrangement of chemicals and solvent (2nd , 3rd and 4th experiment) ..........21

3-3-6

Serial dilution (2nd , 3rd and 4th experiment) .................................................213-3-7

Addition to the cells (2nd , 3rd and 4th experiment) .......................................22

3-3-8

Final constituents of each well of the plate (2nd , 3rd and 4th experiment) ....23

4. Preparation of positive control and negative control .........................................24
4-1

Preparation of cells ...........................................................................................24

4-2

Preparation of 4-nitrobenzyl bromide (4-NBB) and treatment of THP-G8 cells...........................................................................................................................25

4-3

Final constituents of each well of the plate ......................................................27

4-4

Preparation of lactic acid (LA) and treatment of THP-G8 cells ......................27

4-5

Final constituents of each well of the plate ......................................................30

4-4

Measurement ....................................................................................................30

5.

Calculation of the transmittance factors ............................................................31
5-1 Reagents ..............................................................................................................31
5-2 Preparation of luminescence reaction solution ....................................................31
5-3 Bioluminescence measurement ...........................................................................32

6.

Measurement of luciferase activity .....................................................................34

7.

Criteria ..................................................................................................................39
7-1

Definition of the parameters used in the IL-8 Luc assay .................................39

7-2

Acceptance criteria ...........................................................................................39

7-3

The criteria to identify sensitizers in the IL-8 Luc assay .................................40

8.

Update record .......................................................................................................43

9.

Appendix 1

10.

Appendix 2 Validation of reagents and equipment .......................................47

11.

Appendix 3 Calculation of the parameters used in the IL-8 Luc assay ......51

Principal of measurement of luciferase activity ........................45

3

1.

Materials

1-1

Cells

The human macrophage-like cell line THP-1 was obtained from the American Type
Culture Collection (Manassas, VA, USA). A THP-1-derived IL-8 reporter cell line,
THP-G8, that harbors the SLO and SLR luciferase genes under the control of the IL-8
and GAPDH promoters, respectively, was established by the Dept. of Dermatology,
Tohoku University School of Medicine.
(Takahashi T. et al. An in vitro test to screen skin sensitizers using a stable THP-1derived IL-8 reporter cell line, THP-G8. Toxicol Sci, 124(2), 359-369, 2011)(International patent publication No. WO2012/002507A1)
1-2

Reagents and equipment

For maintenance of the THP-G8 cells•

RPMI-1640 (GIBCO Cat#11875-093, 500 mL)•

FBS (Biological Industries Cat#04-001-1E Lot:715004)•

100 X concentrated antibiotic and antimycotic (10,000 U/mL of penicillin G, 10,000µg/mL of streptomycin and 25 µg/mL of amphotericin B in 0.85 % saline) (e.g.,
GIBCO Cat#15240-062)•

G418 (CAS:108321-42-2, Nacalai Tesque Cat#16513-84)•

Puromycin (CAS:58-58-2, InvivoGen Cat#ant-pr-1)

For chemical exposure, positive control, negative control, solvents•

4-Nitrobenzyl bromide (CAS:100-11-8, Aldrich Cat#N13054)•

Lactic acid (CAS:50-21-5, Sigma Cat#L6661)•

X-VIVOTM 15 (Lonza, 04-418Q): Chemically defined, serum-free hematopoietic
cell medium.

For measurement of the luciferase activity•

Tripluc® Luciferase assay reagent (TOYOBO Cat#MRA-301)

4

Expendable supplies•

T-75 flask tissue culture treated (e.g., Corning Cat#353136)•

96-well flat-bottom black plate (for measurement of the luciferase activity, e.g.,
Greiner Bio-one Cat#655090, Nunc Cat#165305)•

96 well assay block, 2 mL (e.g., Costar Cat#3960)•

Reservoir•

PipetteEquipment for measurement of luciferase activity•

Measuring device: a microplate-type luminometer with a multi-color detection
system that can accept an optical filter
e.g.: Phelios AB-2350 (ATTO), ARVO (PerkinElmer), Tristar LB941 (Berthold)•

Optical filter: 600 nm long-pass filter, 600～700 nm band-pass filter•

Measuring time: set at 1～5 sec/well measuring time

Others•

Pipetman•

8 channel or 12 channel pipetman (optimized for 20~100 μL and 0.5~10 μL)•

Plate shaker (for 96 well plate)•

CO2 incubator (37°C, 5% CO2)•

Water bath•

Cell counter: hemocytometer, trypan blue

5

1-3
1-3-1

Culture medium
A medium: for maintenance of THP-G8 cells (500 mL, stored at 2-8°C)
Final

Reagent

Company

Concentration

concentration
in medium

RPMI-1640

GIBCO #11875-093

FBS

Biological Industries
Cat#04-001-1E

Requiredamount--

445 mL

-

10 %

50 mL

100×

1×

5 mL

10 mg/mL

0.15 μg/mL

7.5 μL50 mg/mL

300 μg/mL

3 mL

Lot:715004
Antibiotic ande.g., GIBCO #15240-062

antimycotic
Puromycin (CAS:58-

InvivoGen # ant-pr-1

58-2)
G418 (CAS:108321-

Nacalai tesque #16513-84

42-2)

1-3-2

B medium: for luciferase assay (30 mL, stored at 2-8°C)
Final

Reagent

Company

Concentration

concentration
in medium

RPMI-1640

GIBCO #11875-093

FBS

Biological Industries
Cat#04-001-1E

Requiredamount

-

-

27 mL

-

10 %

3 mL

Lot:715004

1-3-3

C medium: for thawing THP-G8 cells (30 mL, stored at 2-8°C)
Final

Reagent

Company

Concentration

concentration
in medium

RPMI-1640

GIBCO #11875-093

FBS

Biological Industries
Cat#04-001-1E

Requiredamount--26.7 mL

-

10 %

3 mL

100×

1×

0.3 mL

Lot:715004
Antibiotic-Antimycotic

GIBCO #15240-062

6

1-4

Thawing of THP-G8 cells•

Pre-warm 9 mL of C medium in a 15 mL polypropylene conical tube in a 37°C
water bath (for centrifugation) and 15 mL of C medium in a T-75 Flask at 37°C
in a 5% CO2 incubator (for culture).

•

Thaw frozen cells (2x106 cells / 0.5 mL of Freezing Medium) in a 37°C waterbath, then add to a 15 mL polypropylene conical tube containing 9 mL of prewarmed C medium. Centrifuge the tube at 350 x g at room temperature for 5 min,discard the supernatant, and resuspend in 15 mL of pre-warmed C medium in a
T-75 Flask. Cells are incubated at 37°C, 5% CO2.

1-5

Maintenance of THP-G8 cells•

Pre-warm A medium in a T-75 Flask at 37°C in a 5% CO2 incubator. The culture
medium should be changed to A medium 3 or 4 days after thawing. At that time,
count the number of cells, centrifuge the tube at 350 x g for 5 min, discard thesupernatant, and resuspend in pre-warmed A medium in a T-75 Flask. Cells arepassaged at 2～5x105/mL, depending on the condition of the cells, and incubatedat 37°C, 5% CO2.

•

The interval between subcultures should be 3~4 days. Cells can be used between
one and six weeks after thawing.

7

2.

Preparation of cells for assay

A cell passage should be done 2-4 days before the assay.
Use cells between 1 and 6 weeks after thawing.
Pre-warm B medium in a 37°C water bath. Count the number of cells and collect the
number of cells needed (2.5 x 106 cells for one chemical are required, but to have someleeway, 3.75 x 106 cells for one chemical should be prepared), centrifuge the tube at 350
x g, 5 min. Resuspend in pre-warmed B medium at a cell density of 1×106/mL. Transfer
the cell suspension to a reservoir, and add 50 µL of cell suspension to each well of a 96well flat-bottom black plate using an 8 channel or 12 channel pipetman. (cf. Figure 1, row
C-F)
Figure 1.
flatbottom
black
A
B
C

D

E

F
G
H

1

2

3

4

5

6

7

8

9

10

11

12

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul

Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium50ul
Cell
5x10^4
B medium
50ul

8

3.

Preparation of chemicals and cell treatment with chemicals

Prepare chemicals and add them to wells after preparing the cells.
3-1

Dissolution in X-VIVOTM 15 (cf. Figure 2)

Prewarm X-VIVOTM 15 (5 mL) for each chemical at room temperature. Weigh 20 mg ofa test chemical in a microfuge tube (1.5 mL) and add X-VIVOTM 15 up to 1 mL. Vortex
the microcentrifuge tube vigorously. If necessary, sonicate until the chemical iscompletely dispersed.
If the chemical is soluble at 20 mg/mL, dilute the solution 5 times with X-VIVOTM 15,
and transfer 500 µL (1st experiment) or 750 µL (2nd, 3rd and 4th experiments, dilute with
X-VIVOTM 15 if necessary as described on the next page) of the diluted solution to the12th line of a 96 well assay block.
If the chemical is not soluble at 20 mg/mL, shake the microfuge tube on a rotor (e.g.,
WKN-2210, WAKEN B TECH Co. Ltd, Kyoto, Japan) at a maximum speed of 8 rpm for
more than 30 min, until just before centrifugation. After centrifugation at 15,000 rpm(≈20,000 x g) for 5 min, transfer 500 µL (1st experiment) or 750 µL (2nd, 3rd and 4th
experiments, dilute with X-VIVOTM 15 if necessary as described on the next page) of thediluted solution to the 12th line of a 96 well assay block. If undissolved chemical either
precipitates or floats, remove the solution carefully so that no undissolved chemical ispresent in the solution.

9

Figure 2.

In the first experiment (1st experiment), conduct 11 serial dilutions at a common ratioof 2 from the highest concentration using X-VIVOTM 15.
In the second, third and fourth experiments (2nd, 3rd and 4th experiments), determine theminimum concentration at which Inh-GAPLA (mentioned later in 7-1) is lower than 0.05
in the 1st experiment (CV05). Use the concentration one step (2-times) higher than thisdetermined concentration as the highest concentration of the chemical to test, and conduct
11 serial dilutions at a common ratio of 1.5 from this highest concentration. If InhGAPLA does not decrease to less than 0.05 or is less than 0.05 at the highest concentrationin the 1st experiment, conduct 11 serial dilutions at a common ratio of 1.5 from the highest
concentration in the 1st experiment.
For example, in Figure 3 below, the minimum concentration at which Inh-GAPLA isless than 0.05 is 1/128. The highest concentration of the chemical to test is theconcentration one step (2-times) higher than 1/128, which is 1/64.
In Figure 4 below, Inh-GAPLA did not decrease to less than 0.05. In such cases, thehighest concentration of the chemical to test is the highest concentration in the 1st
experiment, namely 1/2.

10Figure 3.

Figure 4113-2

When the chemical is soluble at 20 mg/mL in X-VIVOTM 15

3-2-1

Arrangement of chemicals and vehicle (1st experiment)

Add 500 µL of the 5-fold diluted solution to the #A12 well and 250 µL of X-VIVOTM 15
to wells #A1-#A11 of a 96 well assay block, 2 mL.
3-2-2

Serial dilution (1st experiment)

Conduct 11 serial dilutions at a common ratio of 2 as indicated in Figure 5 from well
#A11 to well #A2. (Transfer 250 µL to the next (left) well.) Make sure not to use blue
pipette tips previously used for dissolving and transferring chemicals in 3.1.
Figure 5.

12

3-2-3

Addition to the cells (1st experiment)

Add 50 μL to THP-G8 in a 96-well flat-bottom black plate using an 8 channel or 12channel pipetman. Shake the plate on a plateshaker, and incubate in a CO2 incubator for
16 hours (37°C, 5% CO2).
Figure 613

3-2-4. Final constituents of each well of the plate. (1st experiment)
Figure 714

3-2-5

Arrangement of chemicals and vehicle (2nd, 3rd and 4th experiment)

Add 750 μL of the X-VIVOTM 15 solution of the chemical prepared at the highest
concentration defined by the 1st experiment (1/A) to well #A12, and 250 μL of X-VIVOTM
15 to wells #A1-#A11of 96 well Assay Block, 2 mL.
3-2-6

Serial dilution (2nd, 3rd and 4th experiment)

Conduct 11 serial dilutions at a common ratio of 1.5 as indicated in Figure 8 from well
#A11 to well #A2. (Transfer 500 μL to the next (left) well) Make sure not to use the blue
tips previously used for dissolving and transferring chemicals in 3.1.
Figure 815

3-2-7

Addition to the cells (2nd, 3rd and 4th experiments)

Add 50 μL to THP-G8 in a 96-well flat-bottom black plate using an 8 channel or 12channel pipetman. Shake the plate on a plateshaker, and incubate in a CO2 incubator for
16 hours (37°C, 5% CO2).
Figure 916

3-2-8. Final constituents of each well of the plate. (2nd, 3rd and 4th experiment)
Figure 10

17

3-3
3-3-1

When the chemical is not soluble at 20 mg/mL in X-VIVOTM 15
Arrangement of chemicals and vehicle (1st experiment)

Add 500 μL of the supernatant to the #A12 and 250 μL of X-VIVOTM 15 to wells #A1#A11of a 96 well Assay Block, 2 mL.
3-3-2

Serial dilution (1st experiment)

Conduct 11 serial dilutions at a common ratio of 2 as indicated in Figure 11 from well
#A11 to well #A2. (Transfer 250 μL to the next (left) well). Make sure not to use the blue
tips previously used for dissolving and transferring chemicals in 3.1.
Figure 11.

18

3-3-3

Addition to the cells (1st experiment)

Add 50 μl to THP-G8 in a 96-well flat-bottom black plate using an 8 channel or 12channel pipetman. Shake the plate on a plateshaker, and incubate in a CO2 incubator for
16 hours (37°C, 5% CO2).
Figure 12.

19

3-3-4. Final constituents of each well of the plate. (1st experiment)
Figure 13.

20

3-3-5

Arrangement of chemicals and vehicle (2nd, 3rd and 4th experiment)

Add 750 μL of the distilled X-VIVOTM 15 solution of the chemical prepared at the highest
concentration defined by the 1st experiment (1/A) to well #A12, and 250 μL of X-VIVOTM
15 to wells #A1-#A11 of 96 well Assay Block, 2 mL.
3-3-6

Serial dilution (2nd, 3rd and 4th experiment)

Conduct 11 serial dilutions at a common ratio of 1.5 as indicated in Figure 14 from well
#A11 to well #A2. Transfer 500 μL to the next (left) well. Make sure not to use the blue
tips previously used for dissolving and transferring chemicals in 3.1.
Figure 14.

21

3-3-7

Addition to the cells (2nd, 3rd and 4th experiments)

Add 50 μL to THP-G8 in a 96-well flat-bottom black plate using an 8 channel or 12channel pipetman. Shake the plate on a plateshaker, and incubate in a CO2 incubator for
16 hours (37°C, 5% CO2).
Figure 15.

22

3-3-8. Final constituents of each well of the plate. (2nd, 3rd and 4th experiments)
Figure 16.

23

4. Preparation of positive control and negative control
4-1

Preparation of cells

Add 50 μL of the cell suspension to wells #C1-#C4, #D1-#D4, #E1-#E4, #F1-#F4 of a
96-well flat-bottom black plate (flat bottom) (cf. Figure 17).
Figure 17.
flatbottom
black

1

2

3

4

Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium

Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium

Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium

Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium
Cell
5x10^450ul medium

5

6

A

B
C
D
E
F
G

H

24

7

8

9

10

11

12

4-2

Preparation of 4-nitrobenzyl bromide (4-NBB) and treatment of THP-G8 cells

20 mg of 4-NBB is prepared in a 1.5-mL microfuge tube, to which X-VIVOTM 15 isadded up to 1 mL. The tube is vortexed vigorously and shaken on a rotor (e.g., WKN2210, WAKEN B TECH Co. Ltd, Kyoto, Japan) at a maximum speed of 8 rpm for at least
30 min. After centrifugation at 20,000g for 5 min, the supernatant is diluted by a factorof 4 with X-VIVOTM 15, and 500 μl of the diluted supernatant is transferred to a well in a
96-well assay block. The diluted supernatant is further diluted with X-VIVOTM 15 atfactors of 2 and 4, and 50 μl of the solution is added to 50 μl of THP-G8 cell suspension
in the wells of a 96-well flat-bottom black plate. (cf. Figure 18, 19) Each concentrationof the positive control should be tested in 4 wells. The plate is agitated on a plateshaker,
and incubated in a CO2 incubator for 16 hours (37°C, 5% CO2)
Figure 18.

25

Figure 19.

26

4-3

Final constituents of each well of the plate

Figure 20.

4-4Preparation of lactic acid (LA) and treatment of THP-G8 cells

20 mg of LA prepared in a 1.5-mL microfuge tube, to which X-VIVOTM 15 is added up
to 1 mL (20 mg/ mL). Twenty mg/mL of LA solution is diluted by a factor of 5 with XVIVOTM 15 (4 mg/mL); 500 μl of this 4 mg/mL LA solution is transferred to a well of a
96-well assay block. This solution is diluted by a factor of 2 with X-VIVOTM 15 and thendiluted again by a factor of 2 to produce 2 mg/mL and 1 mg/mL solutions. 50 μl of these3 solutions and vehicle control (X-VIVOTM 15) are added to THP-G8 in a 96-well flatbottom black plate. (cf. Figure 21, 22) Each concentration of the negative control is tested
in 4 wells. The plate is agitated on a plateshaker and incubated in a CO2 incubator for 16
hours (37°C, 5% CO2).

27

Figure 21.

28

Figure 22.

29

4-5

Final constituents of each well of the plate

Figure 23.

4-6

Measurement

Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature either
in a water bath or at ambient air temperature. Power on the luminometer 30 min before
starting the measurement to allow the photomultiplier to stabilize.
Transfer 100 µL of pre-warmed Tripluc to each well of the plate containing the reference
samples using a pipetman. Shake the plate for 10 min at room temperature (about 20°
C) using a plate shaker. Remove bubbles in the solutions in the wells if they appear. Placethe plate in the luminometer to measure the luciferase activity. Bioluminescence ismeasured for 3 sec each in the absence (F0) and presence (F1) of the optical filter.
Copy the results of the F0 and F1 measurements (values are expressed as counts) andpaste them into the appropriate area in the “Data Input” sheet of the Calculation Sheet.
Calculated factors and graphs will appear on the “4-NBB, Lactic acid” sheet of the
Calculation Sheet. The acceptance criterion is that Ind-IL8LA should be more than 5.0 atleast in one concentration of the positive control, 4-NBB, in each run and that Ind-IL8LA
should be less than 1.4 at any concentration of the negative control, lactic acid, in eachrun. Continue the experiment if this criterion is accepted.

30

5. Determination of the transmission coefficients of optical filter for SLO and SLR
Color discrimination in multi-color reporter assays is generally achieved using
detectors (luminometer and plate reader) equipped with optical filters, such as sharp-cut(long-pass) filters and band-pass filters. The transmittance factors of these filters for eachbio-luminescence signal color must be calibrated prior to all experiments by following
the protocols below.
5-1•

Reagents
Single reference samples:
Lyophilized purified SLO enzyme
Lyophilized purified SLR enzyme•

Assay reagent:
Tripluc® Luciferase assay reagent（TOYOBO Cat#MRA-301）•

B medium: for luciferase assay (30 mL, stored at 2 – 8°C)

5-2

Preparation of luminescence reaction solution

Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature either
in a water bath or at ambient air temperature. Power on the luminometer 30 min before
starting the measurement to allow the photomultiplier to stabilize.
Add 200 µL of 10 ~ 100 mM Tris/HCl or Hepes/NaOH (pH 7.5 ~ 8.0) supplemented
with 10% (w/v) glycerol to each tube of lyophilized purified luciferase sample to dissolve
the enzymes, divide into 10 µL aliquots in 1.5 mL disposable tubes and store in a freezer
at -80°C. The stored frozen solution of the reference samples can be used for up to 6
months.
31

Add 1 mL of B medium to each tube of frozen reference sample (10 µL sample per tube).
Keep the reference samples on ice to prevent deactivation.
5-3

Bioluminescence measurement

Transfer 100 μL of the diluted reference samples to a 96-well flat-bottom black plate as
shown below (the SLO reference sample to #B1, #B2, #B3, the SLR reference sample to#D1, #D2, #D3)..
Figure 24.
1

2

3

SLO

SLO

SLO

1/1

1/1

1/1

SLR

SLR

SLR

1/1

1/1

1/1

4

5

6

7

8

9

10

11

12

Transfer 100 µL of pre-warmed Tripluc to each well of the plate containing thereference sample using a pipetman. Shake the plate for 10 min at room temperature(about 20°C) using a plate shaker. Remove bubbles in the solutions in wells if they
appear. Place the plate in the luminometer to measure the luciferase activity.
Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F1) of
the optical filter. An example of the raw output data is shown below.

32

Figure 25. An example of the raw output data

Transmission coefficient of the optical filter were calculated as follow:
Transmission coefficient (SLO (κOR60))= (#B1 of F1+ #B2 of F1+ #B3 of F1) / (#B1 of
F0+ #B2 of F0+ #B3 of F0)
Transmission coefficient (SLR (κRR60))= (#D1 of F1+ #D2 of F1+ #D3 of F1) / (#D1 of
F0+ #D2 of F0+ #D3 of F0)
In the case shown above,
Transmission coefficient (SLO (κOR60))=(2043+1998+2018) / (9567+9782+9621)=0.21,
Transmission coefficient (SLR (κRR60))=(5749+5765+5784) / (8561+8469+8495)=0.68
Calculated transmittance factors are used for all the measurements executed using
the same luminometer.

33

6.

Measurement of luciferase activity

Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature either
in a water bath or at ambient air temperature. Power on the luminometer 30 min before
starting the measurement to allow the photomultiplier to stabilize.
Transfer 100 µL of pre-warmed Tripluc from the reservoir to each well of the platecontaining the reference sample using an 8 channel or 12 channel pipetman. Shake theplate for 10 min at room temperature (about 20°C) using a plate shaker. Remove bubbles
in the solutions in the wells if they appear. Place the plate in the luminometer to measure
the luciferase activity. Bioluminescence is measured for 3 sec each in the absence (F0)and presence (F1) of the optical filter. In case alternative settings are used, e.g., dependingon the model of luminometer used, these settings should be justified.
The specific procedure for how to manipulate the raw data is shown below using theanalysis of DNCB as an example (Figure 26).
Figure 26. An example of the raw output data (DNCB).

34

1st. Add the information regarding the name of laboratory, the round of experiments if
multiple experimental sets are performed, the experiment number, date, the operator,
chemical code, the highest concentration, dissolved or undissolved in X-VIVOTM 15, andcomments if any to the “FaceSheet” sheet of the Calculation Sheet (Figure 27). In thisCalculation Sheet, the highest soluble concentrations in the next experiments areautomatically shown based on the results.
Figure 27. The “FaceSheet” sheet of the Calculation Sheet.

35

2nd. Copy the results of the F0 and F1 measurements (values are expressed as counts)
and paste them into the appropriate area in the “Data Input” sheet of the Calculation
Sheet shown below (Figure 28). In addition, input the transmittance factors calculated inchapter 5. Calculation of the transmittance factors to TF2 of the “Data Input” sheet(Figure 28).
Figure 28. The “Data Input” sheet of the Calculation Sheet.

36

Next, the calculated results for the parameters of the IL-8 Luc assay for eachconcentration, e.g., IL8LA, GAPLA, nIL8LA, the mean ± SD of IL8LA, the mean ± SD
of GAPLA, the mean ± SD of nIL8LA, the mean ± SD of Ind-IL8LA, the mean ± SD of
Inh-GAPLA, and a graphical presentation with the 95% confidence interval andjudgment, will automatically appear on the “Result Format” sheet and the “Graph” sheetof the Calculation Sheet, respectively (Figure 29, 30).
Figure 29. The “Result Format” sheet of the Calculation Sheet.

37

Figure 30. The “Graph” sheet of the Calculation Sheet.

38

7.

Criteria

7-1

Definition of the parameters used in the IL-8 Luc assay

The way of calculation of these parameters are described in Appendix 3.
Figure 31.
AbbreviationsGAPLA

SLR luciferase activity reflecting GAPDH promoter activity

IL8LA

SLO luciferase activity reflecting IL-8 promoter activity

nIL8LA

IL8LA / GAPLAInd-IL8LA
Inh-GAPLA
CV05

7-2

Definition

nIL8LA of THP-G8 cells treated with chemicals / nIL8LA of untreatedcells
GAPLA of THP-G8 treated with chemicals / GAPLA of untreated cells
Minimum concentration at which chemicals show less than 0.05 of InhGAPLA

Acceptance criteria

The following acceptance criteria should be satisfied when using the IL-8 Luc assay
method.
•In the assay of positive control, Ind-IL8LA should be more than 5.0 at least in one
concentration in each experiment.

•

In the assay of negative control, Ind-IL8LA should be less than 1.4 at any
concentrations in each experiment.

•

Data from plates in which the GAPLA of control wells with cells is less than 5 timesof that of wells without cells should be rejected.

•

Data from plates in which the Inh-GAPLA of all concentrations of the test or control
chemicals is less than 0.05 should be rejected. In this case, the first test should be
repeated from the lowest final concentration of the previous test.
39

7-3

The criteria to identify sensitizers by the IL-8 Luc assay

1. The criterion to determine positive or negative in each experiment:
Chemicals that demonstrate Ind-IL8LA ≥ 1.4 and the lower limit of the 95% confidence
interval of Ind-IL8LA ≥ 1.0 are judged as positive.
The way of calculation of the 95% confidence interval of Ind-IL8LA is described inAppendix 3.
Figure 32.Judgment for each run

THP-G8(IL-8 reporter
cells)

Chemicals

Ind-IL8LA<1.4or
The lower limit of the
95% confidence
interval<1.0

Negative

Ind-IL8LA>=1.4
and
The lower limit of the
95% confidence
interval>=1.0

Positive

2. The criterion to determine sensitiser, supposed non-sensitiser, non-sensitiser orinconclusive
To determine the judgment of the IL-8 Luc assay, the experiments are conducted atmost 4 times. To repeat the experiments, the highest concentration in the 2nd, 3rd, and the
4th experiment is determined as follows.
1st experiment: THP-G8 cells are stimulated with chemicals serially diluted at acommon ratio of 2 using the supernatant or completely dissolved solution.
2nd, 3rd and 4th experiments: Determine the minimum concentration at which InhGAPLA is lower than 0.05 in the 1st experiment. Use the concentration one step (2times) higher than this determined concentration as the highest concentration of thechemical to test, and conduct 11 serial dilutions at a common ratio of 1.5 from thehighest concentration. If Inh-GAPLA does not decrease to 0.05 at any concentrations, or
Inh-GAPLA is lower than 0.05 only at the highest concentration in the 1st experiment,40

conduct 11 serial dilutions at a common ratio of 1.5 from the supernatant or solution.
Based on positive or negative judgment in each experiment, the judgment of the IL-I Luc
assay was determined by the following criterion.
Test chemicals that provide two positive results from among the 1st, 2nd, 3rd or 4th
experiment are identified as sensitisers whereas those that give three negative results fromamong the 1st, 2nd, 3rd or 4th runs are identified as supposed non-sensitisers (Table 32).
Among supposed non-sensitisers, if chemicals are dissolved at 20 mg/ml in X-VOVOTM
15, they are judged as non-sensitisers. If chemicals are not dissolved at 20 mg/ml in XVOVOTM 15, or there is cause for uncertainty regarding the extent of dissolution or
miscibility, chemicals that give less than 0.8 of Inh-GAPLA are judged as non-sensitisers,
while those that give 0.8 or higher of Inh-GAPLA are judged as inconclusive (Figure 33).
Table 32. Criteria for identifying positive and supposed negative
1st run

2nd run

3rd run

4th runFinal prediction

Positive

Positive--

Sensitiser

Negative

Positive-

Sensitiser

Negative

Positive

Sensitiser

Negative

Supposed non-sensitiser

Positive-

Sensitiser

Negative

Positive

Sensitiser

Negative

Supposed non-sensitiser

Positive

Sensitiser

Negative

Supposed non-sensitiser-

Supposed non-sensitiser

Negative

Positive

Negative

Positive
Negative

Figure 33. Prediction model for final judgment

41

42

8. Update record
Ver.025, 2021, May 27
Change the criteria for the proposal of the modification of the prediction model of the
IL-8 Luc assay (OECD TG442E)
Ver.024.2, 2017, March 10
Change the criteria.
Ver.024.1, 2017, Feb 28
Change the acceptance criteria.
Change the name of the parameters.
Add negative control, lactic acid.
Ver.024, 2017, Feb 27
Change rt, 25 to 20.
Ver.023, 2016, Feb 9
Add appendix.
Add Acceptance criteria.
Ver. 022E, 2015, Dec. 31
Change positive control to 4-NBB.
Ver. 021E, 2015, Dec. 21
Add schemes showing data transport and delete the graphical presentation of thecriteria.
Ver. 020E, 2015, July 3
Change preparation of chemicals (When the chemical is soluble at 20 mg/mL in XVIVOTM 15, the chemical is diluted 5-fold) and CoCl2.
Ver. 019E, 2015, April 1
Change vehicle to X-VIVOTM 15
43

Ver. 017E, 2013, Nov. 14 distribution
Modify the criteria
Ver. 016E, 2013, Oct. 29 distribution
Delete the criteria I.I.≤0.80
Ver. 015E, 2012, Nov. 12 distribution
Change preparation of chemicals
Ver. 014E, 2012, Oct. 26 distribution
Change preparation of water-soluble chemicals
Change preparation of CoCl2
Delete the description concerning LPS
Change incubation time (5 hours to 16 hours)
Change the quality control of the equipment
Change the criteria
Ver. 013E, 2012, Aug. 03 distribution
Ver. 012E, 2012, July 12 distribution
Ver. 011E, 2012, June 05 distribution
Ver. 008E, 2011, Dec. 19 distribution
IL-8 Luc assay detailed protocol, 2011, Nov. 11 distribution

44Appendix 1
Principal of measurement of luciferase activity
MultiReporter Assay System -Tripluc- can be used with a microplate-type luminometerwith a multi-color detection system, which can equip an optical filter. (e.g. Phelios AB2350 (ATTO), ARVO (PerkinElmer), Tristar LB941 (Berthold)) The optical filter usedin measurement is 600 ～ 620 nm long or short pass filter, or 600～700 nm band passfilter.
(1) Measurement of two-color luciferases with an optical filter.
This is an example using Phelios AB-2350 (ATTO). This luminometer equips a 600 nm
long pass filter (600 nm LP (Filter 1); R60 HOYA Co.), for splitting SLO and SLR
luminescence.
To determine transmission coefficients of the 600 nm LP, first, using purified SLO andSLR luciferase enzymes, measure i) the intensity of SLO and SLR bioluminescenceintensity without filter (F0), ii) the SLO and SLR bioluminescence intensity that passed
through 600 nm LP (Filter 1), and calculate the transmission coefficients of 600 nm LP
for SLO and SLR listed below.

When the intensity of SLO and SLR in test sample are defined as O and R, respectively,i) the intensity of light without filter (all optical): F0 and ii) the intensity of 600 nm LP(Filter 1) transmitted light are described as below.
F0=O+R
F1=κOR60 x O + κRR60 x R
These formulas can be rephrased as follows
1
1
F0
O
1
�1�=�
� �1�
κOR60
κR R60 R
F1

Then using calculated coefficient factors (κOR60 and κRR60) and measured F0 and F1, you
can calculate O and R-value as follows.
45

−1
1
1
O
F0
1
�1� = �� �1�
κOR60
κR R60R
F1This calculation can be performed using the functions "MININVERSE" and "MMULT"in Microsoft Excel. These calculations are integrated in "IL8_Luc_assay_Data_sheet_20170227_Ver.021.1".

46

Appendix 2
Validation of reagents and equipment1

Measurement of transmittance of optical filter for multicolor measurement
For color discriminations in the multi-color reporter assay, detectors (luminometer andplate reader) are usually equipped with optical filters, such as sharp-cut (long-pass) filtersand band-pass filters. The transmittance factors of these filters for each bioluminescencesignal color have to be calibrated prior to all experiments by following the protocols
below.
1-1

Reagents

・Single reference samples:
Lyophilized luciferase enzyme reagent of SLO
Lyophilized luciferase enzyme reagent of SLR・Assay reagent:
Tripluc® Luciferase assay reagent（TOYOBO Cat#MRA-301）
・B medium: for luciferase assay (30 mL, stored at 2–8°C)

Reagent

Company

RPMI-1640

GIBCO #11875-093

FBS

Biological Industries
Cat#04-001-1E Lot:

Final conc. in

Requiredmediumamount

-

-

27 mL

-

10 %

3 mL

7150041-2
1-2-1

Calibration
Preparation of luminescence reaction solution

Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature bybathing in water or ambient air. Start the luminometer 30 min before starting themeasurement for stabilization of the photomultiplier.
Add 200 μL of 100 mM Tris-HCl (pH8.0) contains 10% glycerol to each tube of the47

lyophilized reference samples to dissolve the enzymes, followed by separating them into
1.5 mL disposable tubes at 10 μL each and storing in a freezer at -80°C. The stored frozen
solution of the reference samples can be used for one half year.
Add 1 mL of B medium to each tube of the frozen reference sample (10 μL in a tube)
and label them as SLO1/1, SLR1/1, and SLO/SLR1/1. Keep the reference samples on ice
to prevent deactivation.
Prepare dilution series of the single reference samples of SLO and SLR as follows.
Dilute 0.3 mL of each 1/1 solution with 0.9 mL of B medium to make SLO1/4 and SLR1/4.
In the same manner, prepare 1/16 and 1/64 solution of each. Keep diluted reference
samples on ice.
1-2-2

Bioluminescence measurement

Transfer 100 μL of the diluted reference samples to a 96-well flat-bottom black plate
as shown below.
Figure 31.
1

2

3

4

5

6

7

8

9

10

11

12

SLO SLO SLO

SLO

SLO

SLO

SLO

SLO

SLO

SLO

SLO

SLO

1/1 1/1

1/1

1/4

1/4

1/4

1/16

1/16

1/16

1/64

1/64

1/64

SLR SLR

SLR

SLR

SLR

SLR

SLR

SLR

SLR

SLR

SLR

SLR

1/1

1/1

1/4

1/4

1/4

1/16

1/16

1/16

1/64

1/64

1/64

A
B
C
D

1/1

E
F
G
H

Transfer 100 μL of pre-warmed Tripluc to each well containing the reference samplesof the plate using a pipetman. Shake the plate for 10 min at room temperature (about
20°C) with a plate shaker. Remove bubbles on the solutions in wells if they appear. Placethe plate into the luminometer of the under-test to measure the luciferase activity.

48

Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F1) of the
optical filter. The example of the raw output data was shown below.
Figure 32.

Copy the results of the F0 and F1 measurement (values are expressed as counts) andpaste it to the appropriate area in the “Data Input” sheet of the Calculation Sheet for data
analyses shown below.
Figure 33.

Record all the results for quality control.

49

2 Quality control of equipment
In order to confirm the detector stability as the quality control, the reference luciferase
sample, optical property, the protocol described hereafter should be performed at the
beginning of the experiments every day using reference LED light source plates equippedwith stabilized LEDs. LED plate data typically fluctuates up to 1.5% (σ). Disagreement
to the old data should be less than 3×σ (= 4.5%). Here describes an example using acommercially available reference light source, TRIANT® (wSL-0001) by ATTO (Tokyo,
Japan).
Start LED plate and select “PMT” mode.
Select three-color (BRG) mode and adjust light intensity to 1/10 (10E-1).
Place the LED plate into the luminometer. Light intensity is measured for 3 sec each in
the absence (F0) and presence (F2) of the optical filter.
Read and record the data at the position of #F6, #E6, and #D6 that are LEDs of blue,
green, and red LEDs, respectively.
LED plate data typically fluctuates up to 1.5% (σ). Disagreement to the old data shouldbe less than 3×σ (= 4.5%).

50

Appendix 3
Calculation of the parameters used in the IL-8 Luc assay
The j-th repetition (j = 1 to 4) of the i-th concentration (j = 0 to 11) is measured for
IL8LA and GAPLA respectively. The normalized IL8LA is referred as nIL8LA, and isdefined as
nIL8LAij = IL8LAij / GAPLAij.
1. Ind-IL8LA
The fold increase of the averaged nIL8LA for the repetition on the i-th
concentration compared with it on the 0 concentration, Ind-IL8LA, is the primarymeasure of this assay. This ratio is able to write by the following formula,
Ind-IL8LAi =｛(1/4)xΣj nIL8LAij｝/｛(1/4)xΣj nIL8LA0j｝.
The 95% confidence interval theorem known as Fieller’s theorem is obtained from thefollowing formula.

 − B − B 2 − 4AC − B + B 2 − 4AC 
,

,
2A
2A


sd 2yisd02
22
where A = x − t 0.975 ( ν ) ×, B = −2 × x × y , C = y i − t 0.975 ( ν ) ×
, and
n0
n yi
2
0

2

x 02 =｛(1/4)xΣj nIL8LA0j｝, sd20 =(1/3)xΣj (nIL8LA0j-x0)2, n 0 =4,

y 02 =｛(1/4)xΣj nIL8LAij｝2, sd2yj =(1/3)xΣj (nIL8LAij-y�i)2, n yi =4,t 0.975(ν ) is 97.5 percentile of the central t distribution with the ν of the degree of

freedom.
51

2. Inh-GAPLAThe Inh-GAPLA is a ratio of the averaged GAPLA for the repetition of the i-th
concentration compared with it of the 0 concentration, and this is written by
Inh-GAPLAi =｛(1/4)xΣj GAPLAij｝/｛(1/4)xΣj GAPLA0j｝

52