Glutathione Fluorescent Detection Kit

Allow the kit reagents to come to room temperature for 30 minutes.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. assay Buffer Prepare the Assay Buffer by diluting the supplied Assay Buffer Concentrate with an equal volume of deionized water.
Mix thoroughly.
Stable at 4 °C for 3 months.
Sample Diluent Prepare the Sample Diluent by diluting one part 5 % SSA 1:5 with four parts diluted Assay Buffer and vortex thoroughly.
The pH of the Sample Diluent must be > 6.
Sample Diluent can be stored at 4 °C for one month.
Standard Preparation GSH Standards are prepared by labeling eight test tubes as #1 through #8.
Briefly vortex to mix and then spin the vial of standard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 450 μL of Sample Diluent into tube #1 and 250 μL into tubes #2 to #8.
Carefully add 50 μL of the Glutathione Standard to tube #1 and vortex completely.
Take 250 μL of the GSH solution in tube #1 and add it to tube #2 and vortex completely.
Repeat this for tubes #3 through #8.
The concentration of GSH in tubes 1 through 8 will be 25, 12.5, 6.25, 3.125, 1.56, 0.781, 0.391 and 0.195 μM.
Use all Standards within 1 hour of preparation.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Sample Diluent Vol (μL) 450 250 250 250 250 250 250 250 addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Vol of addition (μL) 50 250 250 250 250 250 250 250 Final Conc (μM) 25 12.5 6.25 3.125 1.56 0.781 0.391 0.195 ® www.ArborAssays.com 9 Control Preparation (optional) This optional control solution for ensuring complete conversion of GSSG to GSH can be prepared by adding 5 μL of Oxidized Glutathione Control to 245 μL of Sample Diluent. use within 2 hours.
The Control Preparation ensures that the NADPH and Glutathione Reductase system prepared below in the Reaction Mixture section will adequately reduce GSSG to GSH.
If this optional control is run it should yield a value for Total Glutathione of approximately 10 ± 2 μM. thioStar® Detection reagent Allow the ziploc bag to warm completely to room temperature prior to opening and remove the vial of ThioStar Reagent.
Add the volume of DMSO provided to the vial according to the table below.
Vortex thoroughly.
Store any unused reconstituted Detection Reagent at 4 °C in the ziploc pouch with desiccant and use within 2 months.
Kit K006-F1 Kit K006-F5 Vial Part Number C021-1EA, Plastic vial C036-1EA, Glass vial Volume of DMSO to add per vial 1.5 mL 3.5 mL For # of Wells Up to 60 Up to 140 reaction Mixture Prepare the Reaction Mixture by vortexing the vials of Glutathione Reductase and NADPH Concentrates and then diluting one part each NADPH and Glutathione Reductase Concentrates 1:10 into eight parts Assay Buffer.
Vortex thoroughly.
See Table for suitable volumes.
Store any unused Reaction Mixture at 4 °C in an amber vial for no more than 2 days. reaction Mix Dilution table 1/2 Plate one Plate NADPH Concentrate 150 μL 275 μL Glutathione Reductase Concentrate 150 μL 275 μL Assay Buffer 1.2 mL 2.2 mL ® 10 EXPECT ASSAY ARTISTRY ASSAy ProtoCol - Free AnD totAl GSh We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine GSh concentrations. 1.
Use the plate layout sheet on the back page to aid in proper sample and standard identification.
Set plate parameters for a 96-well Corning Costar 3650 plate. 2.
Pipet 50 μL of treated samples, standards or control into wells in the plate. 3.
Pipet 50 μL of Sample Diluent into Zero wells in the plate. 4.
Add 25 μL of the ThioStar Reagent to each well using a repeater pipet. 5.
Gently tap the sides of the plate to ensure adequate mixing of the reagents. 6.
Incubate at room temperature for 15 minutes. 7.
Read the fluorescent signal from each well in a plate reader capable of reading the fluorescent emission at 510 nm with excitation at 370-410 nm.
This data will be used to determine Free GSH concentration. 8.
Add 25 μL of the Reaction Mixture to each of the wells using a repeater pipet. 9.
Gently tap the sides of the plate to ensure adequate mixing of the reagents. 10.
Incubate at room temperature for 15 minutes. 11.
Read the fluorescent emission at 510 nm with excitation at 370-410 nm.
This data will be used to determine Total GSH concentration. total GSh Content only Total GSH content can be determined directly by leaving out steps 5, 6 and 7.

All samples must be treated with the SSA solution prepared on page 6. All of the SSA treated centrifuged supernatants must have their SSA concentration brought down to 1 % SSA by dilution with Assay Buffer. Further dilutions of the sample, using Sample Diluent (see page 9 for preparation), may be necessary to allow the GSH concentration to be measurement in the assay. Detailed instructions follow. All samples and standards must be in Sample Diluent before starting the assay. use all samples within 2 hours of dilution. Whole Blood, Serum, eDtA or heparin Plasma, or urine Thoroughly mix sample with an equal volume of cold 5 % SSA. Incubate for 10 minutes at 4 °C. Centrifuge at 14,000 rpm for 10 minutes at 4 °C. Collect the supernatant. If the supernatent contains particulates, re-centrifuge the supernatant for 15 minutes and collect the clarified second supernatant. Samples can be stored in aliquots at ≤ -70 °C or analyzed immediately. At this point the SSA concentration will be 2.5 %. The supernatant must be diluted 1:2.5 with Assay Buffer by mixing one part with 1.5 parts of Assay Buffer. The SSA concentration will be 1 %. The sample will have been diluted 1:5 at this point. All final dilutions are to be made in Sample Diluent. Treated Whole Blood must be further diluted at least 1:20 for a recommended final dilution of ≥ 1:100. For Treated Plasma and Treated Urine a final dilution of ≥ 1:5 is recommended, but further dilutions in Sample Diluent may be necessary.

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.