Microtiter Plate Assay for the Measurement of Glutathione

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Microtiter Plate Assay for the Measurement of Glutathione
Cell culture, treatment and cell sample preparation
1. Distribute 5 ml of cells into 60-mm culture dishes
2. Incubate the cells for 48 h in CO2 incubator
3. The cells are washed with HBSS and incubated in 5 ml of experimental media with
chemical treatments for 2 h in CO2 incubator
4. Harvest cells using 0.5 ml of trypsin-EDTA to microcentrifuge tubes [2,500 rpm, 5
min, 4 C] and wash the cell pellets with PBS [2,500 rpm, 5 min, 4 C]
5. Resuspend the cell pellets with 70 l of 2.25% (0.09% X 25) SSA solution and lyse
the cells using the three cycles of freezing-thawing (freeze in liquid nitrogen and thaw
in 37 C water bath for 5 min) [ SSA : 5-Sulfosalicylic acid, Sigma S-0640, 2.25%
(w/v) solution in water, store at refrigerator]
6. Centrifugation (14,000 x g, 20 min, 4 C) and take the supernatant to use “Enzymatic
recycling assay” [The extracts may be assayed directly or stored at –70C]
7. Precipitates are redissolved in 100 l of 0.2 M NaOH solution containing 0.1% SDS
and used for Bradford assay (5 L each)
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Dr. Lee’s Lab
Enzymatic recycling assay
1. Reagent :
Stock solution
Buffer (2X) : 60 mM sodium phosphate, 0.6 mM EDTA, pH 7.5
[100 mM Na2HPO4 49 ml + 100 mM NaH2PO4 11 ml + 200 mM EDTA
0.3 ml + DW 40 ml  adjust pH 7.5 using NaOH, refrigerator]
DTNB (Sigma D-8130) : 1.5 mM (10X) in 0.5% NaHCO3 [dark, refrigerator]
NADPH (Sigma N-1630) : 2 mM (10X) in water [dark, freezer]
Glutathione reductase (Sigma G-3664) : 1.9 U / l
Glutathione, oxidized (GSSH) (Sigma G-4376) : 2 mol/ml [2 mM, 1.2 mg/ml
Buffer (1X)]  1/100 dilute, 20 nmol/ml [20 M, 800 pmol/40 l]
Standard (/40 l) : 0, 6.25, 12.5, 25, 50, 100, 200 pmol
Preparation of reaction mixture (master mix) : mix just before assay
Volume (l)
Final concentration
Distilled water
30
-
Buffer (2X)
50
1X
1.5 mM DTNB
10
0.15 mM
2 mM NADPH
10
0.2 mM
0.05
1.0 U/ml
1.9 U/l GSH reducatse
Total (per well of 96-well plate) 100
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2. Reaction : total reaction volume – 150 l per each well of a 96-well plate
1) Dispense standards and samples (duplicate, final SSA conc.: 0.09%) on ice
Standards : Buffer(1X) containing GSSG, 40 l + 0.45% SSA, 10 l
Samples : Buffer (1X), 48 l + each sample 2 l
2) Add 100 l of master mix prepared above to all each well on ice
3) Measure absorbances at 405 nm using plate reader
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Dr. Lee’s Lab