Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
培养的小胶质细胞裂解液中谷胱甘肽的荧光法估测
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2304 浏览次数: 14282
评审: Xi FengAmey G RedkarPooja Mehta
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参见作者原研究论文
The authors used this protocol in:
Jul 2016
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Abstract
Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent. O-phthalaldehyde has an ability to react specifically with GSH at pH 8 and GSSG at pH 12 respectively. N-ethylmaleimide (NEM) has been used to prevent auto-oxidation of GSH during measurement of GSSG in the present protocol. The original protocol by Hissin and Hilf was developed for glutathione estimation in Rat liver tissue. The present protocol has been standardized following Hissin and Hilf (1976) for the estimation of glutathione in cultured microglial cell lysate but it can also be used for other mammalian cell lysate. In our lab same protocol has been used for the estimation of glutathione in the whole cell lysate of murine neuroblastoma cell, N2a.
Keywords: Glutathione (谷胱甘肽)
Antioxidant (抗氧化剂)
Free radical (自由基)
Microglia (N9) (小胶质细胞（N9）)
o-Phthalaldehyde (邻苯二甲醛)
N-ethylmaleimide (N—乙基马来酰亚胺)
Background
This method was published by Hissin and Hilf in analytical biochemistry way back in 1976 (Hissin and Hilf, 1976). There are methods available to detect GSH accurately however; due to readily oxidative conversion of GSH into GSSG most of the methods give an overestimate of GSSG. Cohen and Lyle (1966) solved the problem by using NEM to prevent oxidative conversion of GSH into GSSG and also preventing GSH to react with OPT during GSSG estimation (Figures 1 and 2). We have used this simple and reliable method to detect GSH and GSSG in our experimental system (microglial cell lysate). The main advantage of this protocol is that, it does not involve sophisticated instrument like high performance liquid chromatography (HPLC) which also needs sufficient expertise to handle as compared to plate reader which is more commonly available and easy to operate (Rahman et al., 2006). 
Figure 1. Schematic of chemical reaction during GSH estimation in the N9 cell lysate
Figure 2. Schematic of chemical reaction during GSSG estimation in the N9 cell lysate
Materials and Reagents
- Pipette tips (Corning, Axygen®, catalog number: T-1005-WB-C-L )
- 0.22 μm filter
- 6 well culture plate (SRL LIFE SCIENCES, catalog number: 30006 )
- Culture flask
- 1.5 ml centrifuge tube (Corning, Axygen®, catalog number: MCT-150-R )
- 0.5 ml tube (Corning, Axygen®, catalog number: 14-222-292 )
- 96 well plate (SPL life sciences, catalog number: 30096 )
- Disposable plastic cell scraper (SRL LIFE SCIENCES, catalog number: 90020 )
- Cell line: In the present protocol mouse microglial cell line, N9 has been used, which was kindly gifted by Dr. Anirban Basu, National Brain Research Centre (NBRC), India (Singh et al., 2016). N9 cell line was developed by retroviral transfection of primary microglia cells with the v-myc or v-mil oncogenes of the avian retrovirus MH2. Cells were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin in a 5% CO2 incubator at 37 °C
- DMEM/F12 (Sigma-Aldrich, catalog number: 56498C )
- Sodium bicarbonate
- Double distilled water
- Fetal bovine serum (FBS) (Genetix Biotech, Cell cloneTM, catalog number: CCS-500-SA-U )
- Penicillin-streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
- Bradford reagent (Bio-Rad Laboratories, catalog number: 5000006 )
- Tri-chloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
- o-Phthalaldehyde (Sigma-Aldrich, catalog number: P1378 )
- N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876 )
- Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251 )
- Glutathione oxidized (GSSG) (Sigma-Aldrich, catalog number: G4376 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
- Dipotassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
- Potassium phosphate dibasic trihydrate (K2HPO4·3H2O)
- EDTA disodium salt (Sigma-Aldrich, catalog number: E5513 )
Note: This product has been discontinued. - 100% ethanol (Merck, catalog number: 1009831011 )
- Methanol (SRL Laboratories, catalog number. 65524 )
- 0.1 M potassium phosphate EDTA buffer (KPE buffer) (see Recipes)
- 50% trichloroacetic acid (see Recipes)
- o-Phthaldehyde solution (10 mg/ml) (see Recipes)
- 0.4 M N-ethylmaleimide (see Recipes)
- 0.1 N sodium hydroxide (see Recipes)
Equipment
- 1 ml and 200 µl pipettes (Eppendorf)
- Table top centrifuge (Sigma-zentrifuges, model: Sigma 3-18KS )
- Microplate reader (BMG LABTECH, model: FLUOstar Omega )
- CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CycleTM CO2 Incubators )
- Sonicator (Sonics & Materials, model: VC 505 )
Software
- Mars data analysis software, ver. 1.01 (Data analysis software for Microplate reader)
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Singh, V., Gera, R., Purohit, M. P., Patnaik, S. and Ghosh, D. (2017). Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate. Bio-protocol 7(11): e2304. DOI: 10.21769/BioProtoc.2304.
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分类
癌症生物学 > 癌症生物化学 > 蛋白质
生物化学 > 蛋白质 > 定量
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