Primary mouse hepatocytes were isolated as described in Supplementary Methods section. Hepatocytes and fibroblasts from NPC patients were pretreated with GSH-EE (5 mM) or N-acetylcysteine (NAC, 10 mM) and then treated with hydrogen peroxide (H2O2, 1 mM) (Sigma) to evaluate cell viability. In addition, 7-days old Npc1-/- mice were treated with 1.25 mmol/kg GSH-EE (Sigma, St. Louis, MO), 2.5 mmol/Kg NAC intraperioteneally (i.p.) or vehicle (saline) every 12 h for 6 weeks to measure mGSH and total GSH levels. For survival studies, 7-days old Npc1-/- mice were treated with GSH-EE, NAC or vehicle every 12 h measuring body weight weekly until demise.
Gsh ee
Manufactured by Merck Group
Sourced in United States
The GSH-EE is a laboratory equipment designed to measure the concentration of glutathione (GSH) in biological samples. It functions by performing a colorimetric assay to quantify the level of reduced glutathione present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Hbm msc, by Lonza (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Raw 264, by Korean Cell Line Bank (1 mentions)
γ glucys, by Merck Group (1 mentions)
Anti cox 4, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti xbp1, by Cell Signaling Technology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
Anti atf4, by Cell Signaling Technology (1 mentions)
Anti grp78, by Cell Signaling Technology (1 mentions)
Anti puma, by Cell Signaling Technology (1 mentions)
Genomic dna kit, by Promega (1 mentions)
Anti atf3, by Cell Signaling Technology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti gadd45a, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
6 protocols using gsh ee
1
Evaluating Oxidative Stress in NPC
2
Pharmacological Replenishment of Glutathione
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glutathione-reduced ethyl ester (GSH-EE; Sigma-Aldrich #G1404) was used for pharmacological replenishment of GSH in WT and AS cultured NPCs. A stock solution of 100 mM in sterile water was prepared (stored at −20 °C) and then further diluted to 0.5 mM working solution (final con.) in a complete embryonic NeuroCult™ Proliferation medium before the treatment. For GSH-EE treatment, the medium was replaced by the working solution for 48 h incubation. As a control, for the separate cohorts of WT and AS vehicle NPCs, the medium was replaced with fresh medium without the supplement of GSH-EE.
3
Cell Culture and Differentiation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa and RAW264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and the American Type Culture Collection (ATCC; Manassas, VA), respectively. The cells were grown in phenol red-free DMEM (Welgene, LM 001-10; Gyeongsan, Korea) supplemented with 10% heat-inactivated fetal bovine serum (HyClone; GE Healthcare, Melbourne, VIC, Australia), 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 2 mM glutamine. hES-MSCs were kindly provided by Hyung-Min Chung (Konkuk University, Korea). Cellular GSH of hES-MSCs was depleted by treating cells with 80 μM BSO (Sigma-Aldrich, St. Louis, MO) for 24 hr and was restored with 1 mM GSH-EE (Sigma-Aldrich) for 2 hr, followed by their functional assays. hBM-MSCs (Lonza, Walkersville, MD) were cultured according to the manufacturer's instructions. Culture, EB formation, and in vitro neuronal differentiation of murine E14TG2a ESCs (ATCC) were performed as previously described (Heo et al., 2017 (link)).
4
Intracellular GSH Quantification Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (40,000 per well, duplicate per condition) were seeded in 96-well plates at 37 °C/5% CO2 in DMEM supplemented or not with different compounds (3 mM GSH, 10 mM GSH, 3 mM GSH + 100 μM β-ME, 3 mM GSH ethylester (GSHEE, Sigma-Aldrich, St. Louis, CA, USA), 1 mM N-acetylcysteine (NAC, Sigma-Aldrich, St. Louis, CA, USA), 150 μM gamma-glutamylcysteine (γ-GluCys, Sigma-Aldrich, St. Louis, CA, USA)). Intracellular GSH level was determined after 24 and 48 h by SAFAS Xenius XOF (Safas) using the GSH fluorimetric assay kit (CS1020, Sigma-Aldrich, St. Louis, CA, USA) according to the manufacturer’s instructions. Relative GSH content was normalized to the number of cells.
5
Antibody Validation for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sulforhodamine B, tunicamycin, and GSH-EE were purchased from Sigma‒Aldrich (St. Louis, MO, USA). A genomic DNA kit was obtained from Promega (Madison, WI, USA). The anti-β-Tubulin (#2128), anti-ATF3 (#18665), anti-GADD45A (#4632), anti-ATF6 (#65880), anti-CHOP (#2895), anti-GRP78 (#3177), anti-XBP1 (#40435), anti-IRE1α (#3294), anti-DR5 (#8074), anti-Bax (#2772), anti-ATF4 (#11815), anti-Puma (#4976), anti-PERK (#3192), anti-p-eIF2α (#9721), anti-eIF2α (#9722), anti-p-JNK (#9251), anti-JNK (#9252), anti-cytochrome c (#4272), anti-COXIV (#4850), anti-Calnexin (#2679), anti-PARP1 (#9542), anti-Poly/Mono-ADP Ribose (#83732), anti-Caspase 3 (#9662), anti-Caspase 8 (#9746), and anti-Caspase 9 (#9502) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The anti-PARP16 antibody (ab154510) was obtained from Abcam (Cambridge, UK), the anti-Sestrin2 antibody (10795-1-AP) was obtained from Proteintech (Rosemont, IL, USA), the anti-CYB5R3 antibody (BS-12162R) was obtained from Bioss Antibodies Inc. (Woburn, MA, USA), the anti-GAPDH antibody (LF-P-A0212) was obtained from AbFrontier (Seoul, Korea), the anti-ARTC1 (SAB1300652) and anti-Flag (F1804) antibodies were obtained from Sigma‒Aldrich, and the mouse anti-FITC (sc-2010) and rabbit anti-rhodamine (sc-2492) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
6
Murine OVA-Induced Allergic Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OVA and GSH-EE were purchased from Sigma Chemical Company (Beijing, China). BALB/c mice were provided by Animal Laboratories of Chinese PLA general hospital (Beijing, China). Protein and enzyme quantification kits were obtained from Jiancheng Biology Company (Nanjing, China). All other chemicals and instruments were obtained from local companies.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!