Cellular Glutathione Detection Assay Kit #13859

#13859 Cellular Glutathione Detection Assay Kit Protocol

A. Solutions and Reagents

1. Reduced Glutathione Standard: Reconstitute lyophilized product in 100 µl of Tris Assay Buffer to create a 100 mM solution.
2. Glutathione-S-Transferase: Reconstitute lyophilized product with 110 µl of Tris Assay Buffer.
3. Tris Assay Buffer: Bring to room temperature before use.
4. Digitonin Lysis Buffer: Thaw and keep on ice while using.
5. Monochlorobimane: Reconstitute lyophilized product with 44 µl of DMSO.

Reduced Glutathione Working Solution as follows (see Table 1).

Table 1: GSH Working Solution for one 96-well plate (100 assays)

NOTE: To create a GSH standard curve, prepare a 100 µM GSH Standard Solution by diluting the 100 mM Reduced Glutathione Standard 1:1000 in Tris Assay Buffer. Run the standard curve in triplicate using 50 µl of standard per well. Create a five point standard curve by starting at 100 µM and using a 1:2 serial dilution in Tris Assay Buffer. Include a blank control. The GSH Standard Solution is not stable at this concentration and should be used within 2 hours or discarded.

Additional Reagents (not supplied): DMSO ( #12611)

B. Preparing Cell Extracts

1. Preparation of cell extracts (10 cm dish)
   1. Apply desired treatment protocol and prepare a control sample without treatment.
   2. Following treatment, aspirate media and add 1.0 ml Digitonin Lysis Buffer to each sample, adjusting Digitonin Lysis Buffer volume when using a small surface area vessel.

      NOTE: If using suspended or detached cells, collect and centrifuge the cell suspension for 5 min at 1,200 rpm. Discard the supernatant and add Digitonin Lysis Buffer to the cell pellet.

   3. Incubate the cell lysates on ice for 5-10 min.
   4. Scrape cells off the plate and transfer suspension to a microcentrifuge tube.
   5. Centrifuge cell lysates for 10 min at 14,000 rpm at 4°C.
   6. Collect the supernatant and run assays or store at -80°C for later use.

      NOTE: use a 10 kDa MWCO centrifugal filter and follow the manufacturer’s instructions for cells that require an additional filtration step.

2. Preparation of cell extracts from 96-well assay plate
   1. Following treatment, centrifuge assay plate for 10 min at 1,200 rpm and carefully remove the supernatant.
   2. Add 50 µl Digitonin Lysis Buffer to each well and incubate at room temperature on shaker for 15 min.
   3. Transfer samples into microcentrifuge tubes and centrifuge samples for 10 min at 14,000 rpm at 4°C.
   4. Collect the supernatants and immediately proceed to GSH assay.

C. Test Procedure

1. Detection of Reduced Glutathione in Cell Extracts
1. Mix Tris Assay Buffer with test sample, GSH standard, and Working Solution in a black 96-well plate with a clear bottom to final volume of 100 µl (See table 2).

Table 2: Detection and Standard Curve Assays

Sample	Test Sample (µl)	GSH Standard (µl)	Working Solution (µl)	Assay Buffer (µl)	Total Volume (µl)
Blank	-	-	50	50	100
Standard	-	50	50	-	100
Sample	1-50	-	50	50 minus sample volume	100



2. Incubate the assay plate at room temperature for 60 min, protected from light.
3. Read plate with a plate reader at an excitation wavelength around 380 nm and emission wavelength of 485 nm.
2. Detection of Reduced Glutathione (GSH) in Live Cells

   The following procedure measures reduced glutathione in live cells using a 96-well plate. The same protocol can be modified for fluorescent imaging and flow cytometry detection.

   1. Culture adherent or suspension cells in a 96-well black plate with clear bottom in duplicate for minimum of 16 hr. Determine the background reading by using at least 2 wells containing growth media without cells.

      NOTE: For best results, a cell number titration is recommended to determine the optimal cell seeding density. We recommend not exceeding 1x10⁵ cells per well.

   2. Create a Live Cell Staining Working Solution by diluting the reconstituted Monochlorobimane stock solution 1:50 in Tris Assay Buffer. For example, dilute 20 µl of Monochlorobimane stock solution in 1 ml Tris Assay Buffer to make 1 ml Live Cell Staining Working Solution, which is enough for one 96-well plate. Mix reagents by gently tapping each side of the plate.
   3. Add 10 µl of Live Cell Staining Working Solution to each well containing 100 µl of growth media.
   4. Incubate samples in a CO₂ incubator for a minimum of 30 min. For best results, measure the fluorescence intensity every 30 min for up to 3 hr or until the signal has reached a plateau.