Homogeneous Add-Mix-Read Assay for Total and Oxidized Glutathione
John Shultz, Mary Sobol, Nancy Murphy, Jolanta Vidugiris; Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 www.promega.com
February, 2011
Assay sensitivity and glutathione levels in various cell lines
Abstract
Lyse
Cells
O
Reduce GSSG
[add DTT]
O
S
N
N
S
S
COOH
1.6
20.4
8.0
HepG2
10,000
45.7
33.3
0.5
7.9
HepaRG
50,000
11.9
6.5
0.3
1.4
A549
5,000
111.6
86.2
1.2
6.6
Rat hepatocytes
20,000
31.2
25.5
1.5
8.6
HO
N
S
S
Cell Line
Luciferin Generation Reagent (GST)
Cells/well
Incubate @ RT 10 min.
Measure Luminescence
120
100
80
60
100
80
60
40
40
20
20
0
0
0
50
100
150
200
250
0
50
GSH
GSSG
16,000
Ratio GSH/GSSS
Vehicle
Menadione
ATP
GSH
GSSG
GSH
10,000
8,000
HepG2
10,000
92.6
2.2
HepaRG®
50,000
35.3
2.5
4,000
A549
5,000
87.9
10.9
2,000
Rat hepatocytes
50,000
19.8
1.1
treated
2,000,000
1,500,000
1,000,000
500,000
GSSG
250
ATP
1.56 3.13 6.25 12.5 25
50
100
treated
vehicle
Extracellular
treated
vehicle
Math Total
treated
vehicle
Combined
A549 adherent lung carcinoma
cells treated with vehicle or 40µM
menadione for 1hr in 96-well
format. The treatment media was
removed and the levels of GSH
and GSSG were determined for
cells (Intracellular) and media
(Extracellular). The Math Total
represents addition of intracellular
and extracellular values.
Combined represents glutathione
values for treated and untreated
cells in suspension without media
removal. Although the total and
oxidized glutathione level in the
media is different than in the cell,
the effect of menadione on the
glutathione pools is still apparent.
Summary
GSH/GSSG-Glo™ allows rapid, direct measurement of total and oxidized
glutathione in cell-culture wells using low cell density.
80
70
The simple protocol eliminates time-consuming sample deproteination and
centrifugation steps required in conventional assays.
60
50
40
The assay can be performed directly in cell culture plates, eliminating sample
transfer problems.
30
20
10
0
0
vehicle
Intracellular
GSH/GSSG ratio
GSSG response
6,000
0
Measuring the effect of compounds on GSSG levels in cells
Net RLU
200
CDNB [µM]
14,000
2.2
0
150
Although both EA (ethacrynic acid) and CDNB (chlorodinitrobenzene) form GSH adducts in the cell,
their effects on cellular ATP and GSSG levels are quite different. Hep G2 cells were treated with doses
of the compounds for 1hr. The values for the control (no compound) was set to 100% and the value s
for the cells exposed to the various concentrations of the compounds was determined and is reported
relative to those values.
63.8
2,500,000
100
EA [µM]
5,000
µM menadione
Luciferin Detection Reagent (LDR)
120
Measuring Intracellular and Extracellular Glutathione
Cells treated directly in tissue culture wells with 40µM menadione or vehicle (0.1% DMSO ).
One set of wells for GSH and one
set of wells for GSSG
Incubate @ RT 5 min.
140
HeLa
Luciferase,
ATP (LDR)
Suspension cells
Measures intracellular
and extracellular
Total Glutathione Lysis Reagent or Oxidized
glutathione
Glutathione Lysis Reagent
140
12,000
Light
Cell Treatment (test compounds)
160
Cells treated directly in tissue culture wells with 40µM menadione or 0.1%
DMSO. Menadione interferes with the mitochondrial electron transport chain
causing an increase in cellular GSSG through the production of reactive
oxygen species. GSH and GSSG concentrations(nmol/106 cells) were derived
from RLU values by comparison to a GSH standard curve.
(Net RLU GSH-Net RLU GSSG)
(Net GSSG/2)
Reduce GSSG
[add DTT]
N
160
% control
85.4
180
Ratio GSH/GSSG = µM GSH - (µM GSSG x 2) / µM GSSG
Ratio GSH/GSSG = RLU GSH - RLU GSSG / RLU GSSG/2
Adherent cells or cells in
suspension
Incubate @ RT 30 min.
103.6
0.6
COOH
Simple add-mix-read assay in the cell culture wells
Remove media (optional)
treated
200
180
The ratio is calculated directly from RLUs or after conversion of RLUs to concentration glutathione using
the standard curve.
GS-R
Block GSH
[Add NEM]
vehicle
28.2
10
The limit of detection was determined in
96-well format using reduced and
oxidized glutathione standards. LOD
GSSG= 0.5nM. LOD GSH = 2.5nM.
Total glutathione and GSSG are measured in separate reactions. For total glutathione measurement all
the glutathione is converted to the reduced form followed by a GST catalyzed reaction to generate one
luciferin for each GSH in the sample. The luciferin is measured using a luciferase reaction. For oxidized
glutathione measurement (GSSG), free sulfhydral groups are blocked with NEM, followed by DTT
addition to convert the GSSG to GSH which is then measured using the GST/luciferase reaction.
Incubate X min.
1
treated
46.2
GSH or GSSG, (µM)
GST
(optional)
Adherent cells
Measures intracellular
glutathione
0.1
vehicle
5,000
1
0.01
nmol/106 cells
HepG2
10
0.001
nmol/106 cells
% control
100
5,000
CH3
Lyse Cells
Remove
media
HeLa
O
Oxidized Glutathione
Grow
cells
1000
GSH
(optional)
Treat
GSH
NO2
Remove
media
Grow
cells
Cells/well
GSSG
GSH/GSSG ratios
Total Glutathione
Treat
Cell line
GSSG
200
Net RLU, thousands
GSH/GSSG-Glo™ assay principle
10000
GSH
CDNB with Hep G2 Cells
Ethacrynic Acid with Hep G2 Cells
100000
S/N
A wide variety of compounds can induce intracellular formation of reactive oxygen species
(ROS) and the resulting cellular damage can eventually lead to apoptosis or necrosis.
Measuring the ratio of reduced to oxidized glutathione (GSH/GSSG ratio) is a way to
determine how such compounds alter the redox potential in the cell. A new method is
presented for rapid determination of the GSH/GSSG ratio in mammalian cells in multi-well
tissue culture plates. The method is based on the formation of luciferin from a glutathione Stransferase catalyzed reaction using GSH and a luciferin precursor. The method utilizes a cell
lysate generated directly in the wells of the cell culture plate and does not require protein
removal steps. In the reaction to measure total glutathione, DTT is added to convert all the
glutathione in the reaction to the reduced form. In the reaction to measure oxidized
glutathione (GSSG), free sulfhydral groups are inactivated with NEM leaving the GSSG intact.
The inactivation step is followed by a reducing step that converts the GSSG to GSH for
quantification in the luminescent reaction. Since the reaction of free sulfhydral groups takes
place immediately upon cell lysis, additional formation of GSSG from air oxidation of GSH is
prevented. The assay takes less than two hours from the time a plate of cells is ready for
measurement and can be performed using less than 1000 cells per well. The performance of
the method is demonstrated by following the effects of compounds such as menadione,
ethacrynic acid and chlorodinitrobenzene on the levels of GSH and GSSG in cells. This is a
fast, easy, and sensitive assay for identification of compounds that alter the GSH/GSSG ratio.
Effect of Compounds on Reduced and Oxidized Glutathione
0
1.5625 3.125 6.25
12.5
25
50
µM menadione
A549 lung carcinoma cells (5,000 cells/well) were exposed for 60 minutes to menadione doses in
Hank’s Balances Salt Solution. A concentration-dependent increase in GSSG was measured and the
decrease in the GSH to GSSG ratio was graphed.
100
The assay measures GSSG directly rather than calculating GSSG from the
difference between reduced glutathione and total glutathione.
Media removal before performing the assay may not be required depending on the
particular media and incubation conditions.