GSH reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate 2-nitro-5-thiobenzoic acid, which exhibits maximal absorbance at 412 nm. Existing reduced glutathione in the sample is inhibited by 2-vinylpyridine. GSSG is then reduced to GSH by glutathione reductase, allowing the quantification of oxidized glutathione.

Taking 50T/48S packing for example:

Note: Perform pretests with 2–3 representative samples prior to formal measurement.

1. Preparation of Lab Instruments

Analytical balance, Mortar and pestle or homogenizer, Visible spectrophotometer, Refrigerated centrifuge, Water bath, 1 mL glass cuvettes, Distilled water

2. Sample Preparation

1)Tissue samples

Rinse fresh tissue twice with PBS. Weigh 0.1g of tissue and place in a homogenizer pre-rinsed with CB0168V-A and pre-cooled on ice. Add 1 mL CB0168V-A (maintain tissue/CB0168V-A ratio), homogenize thoroughly on ice (liquid nitrogen improves efficiency). Centrifuge at 8000 g, 4 °C for 10 min. Collect the supernatant and store at 4 °C until assay. If delayed, store at –80 °C (up to 10 days).

2)Cells

Collect ≥1×10^6 cells, wash twice with PBS (resuspend, 600 g, 10 min), resuspend in 3× cell pellet volume of CB0168V-A. Freeze-thaw 2–3 times (liquid nitrogen and 37 °C water bath). Centrifuge at 8000 g, 4 °C for 10 min, collect supernatant for assay, or store at –80 °C (up to 10 days).

3)Blood samples

Plasma: Centrifuge anticoagulated blood at 600 g, 4 °C, 10 min. Collect plasma, add equal volume CB0168V-A, centrifuge at 8000 g, 4 °C for 10 min. Collect supernatant for assay or store at –80 °C.

Blood cells: Centrifuge anticoagulated blood at 600 g, 4 °C, 10 min. Remove plasma, wash cells 3× with 3× volume PBS. Add equal volume CB0168V-A, mix and incubate at 4 °C for 10 min, centrifuge at 8000 g, 4 °C for 10 min. Collect supernatant for assay or store at –80 °C.

3. Assay Procedure

1)Preheat spectrophotometer for 30 min, set wavelength to 412 nm, and zero with distilled water.

2)Incubate CB0168V-B in water bath at 25 °C (general) or 37 °C (mammals) for 30 min.

3)Prepare standards: dissolve standard powder in 1 mL distilled water (1 mg/mL). Dilute to 15, 10, 5, 2.5, 1.25, and 0 µg/mL (after 10× dilution with CB0168V-A).

4)In EP tubes, add reagents as follows:

4. Calculation of GSSG Content

Generate standard curve (ΔA_standard vs. concentration). Determine sample concentration (y, µg/mL) from ΔA_sample.

By protein concentration:

GSSG(μg / mg prot)=y×V1 ÷(V1 × Cpr)=y÷Cpr

By sample mass:

GSSG(μg /g)= y×V1 ÷(V1 ÷ V2 × W)= y÷W

By cell number:

GSSG(μg /10^6 cell)= y×V1 ÷(V1 ÷ V2 × CN )= y ÷ CN

By liquid volume:

GSSG (µg/mL)=2y

Note:

V2: total supernatant volume (1 mL)

V1: supernatant volume in reaction (100 µL = 0.1 mL)

W: sample weight (g)

Cpr: supernatant protein concentration (mg/mL)

CN: Cell number, in units of 10^6 cells

2: plasma/blood cell samples diluted 2×

1.CB0130S-A contains a protein precipitant; therefore, the supernatant cannot be used for protein concentration assays. If protein content needs to be measured, a separate tissue sample must be prepared.

2.Sample processing requires complete homogenization. If the measurement cannot be completed on the same day, store at -80°C for no more than 10 days.

3.If the measured absorbance exceeds the linear range, increase the sample volume or dilute the sample before testing.

4.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

5.Please wear a lab coat and disposable gloves.

• CB0168V-Instruction Manual(365 KB)