1. Q: What would you suggest to prepare the cell samples for this assay?

Answer: All the detergents in various lysis buffers will cause background noise for the assay. The traditional way to do it is the "freeze and thaw" method (i.e. freeze for 20-30 minutes at -80 C then thaw at room temperature, 2-3 times), but it is too tedious. We found that 0.2% SDS gives the best results among all the detergents tested.

2. Q: Does this Kit work for plant samples? Could you recommend a special protocol to measure gluthatione in plants?

Answer: As long as the sampl  chop 2 g of plant parts (leaf, root, grain, etc.) and ground it in a homogenizer for 2 min with 5-10 ml of 0.1 N HCl; centrifuge to pbtain the  GSH containing supernatant, then adjust pH to 6.5 for the GSH assay.

3. Q: My sample is frozen brain tissue, how should I prepare it for the saasy?

Answer: It is better use PBS, and simply homogenize it, then use the supernatant.

4. Q: Does this kit measure GSSG and GSH directly?

Answer: This kit can be used for measuring total and the reduced form (GSH) directly, the oxidized form (GSSG) is calculated [the total minus GSH]. The GSSG probe is a reducing agent that reduces GSSG to GSH (in order to measure total GSH). The detection limit is around 0.1~0.3 uM.

5. Q: I work with human primary cells. How many cells should I use in the assay?

Answer: Suggest to start with 10,000 cells/well first.

6. Q: Is any of the components in this kit from animals?

Answer: No. The only enzyme (in Component E) is from Baker's Yeast (S. cerevisiae).

7. Q: For the fluorescent detection, an emission filter with 520 nm is recommended. But we have only 535 nm filter. Would it work for this measurement?

Answer: Yes.

8. Q: We don't have the filters for 490/520 (nm). What we have are for 395/535 (nm). Would it work?

Answer: No. the emission is OK, but the excitation is not. The lowest excitation wavelength is at 460 nm.

9. Q: I've tried this kit, but it didn't well for my samples. Here is the protocol I used for obtaining cell culture lysates. Could you take a look and give your suggestion?

Answer:

    - remove supernatant, then wash with HEPES-BSS;[Fine]
    - trypsinize for 2-6 min, stop trypsination;[We suggest to avoid trypsin and try using a rubber scrapper or EDTA to harvest cells, then centrifuge the cells and use the supernatant]
    - centrifuge pellet down at 180g, 5min, 4°C, then wash with 5ml cold PBS; count cells; [Fine]
    - centrifuge pellet down at 180g, 5min, 4°C; [May need to use more cells since you indicated the signal in the undiluted sample is very low.]
    - wash with 5ml COLD PBS; resuspend in ice-cold 5% MPA;[Fine. 5% MPA could be added to the supernatant for deproteination.]
    - mix thoroughly, homogenize by sonication (2x 5 cycles), then centrifuge at 12.00rpm for 5min at 4°C;[Fine]
    - collect supernatant for GSH assay; store on ice OR at -80°C. [Need to neutralize the MPA to pH 4~6, then analyze the undiluted sample with the kit]