γ glutamyl glutamate

Total intracellular GSH (combination of all GSH + GSSG present) levels in the cells were assessed using HPLC as described in Jones and Liang (2009) (link). In brief, cells were plated and after reaching 80% confluency, exposed to AgNP (50 μg/ml) for 24 h. After washing with ice-cold PBS, cells were re-suspended in 500 μl of a solution of 5% perchloric acid, 0.2 M boric acid, and 10 μM γ-glutamyl glutamate (internal standard; Acros Organics; Geel, Belgium). Each sample was briefly sonicated and centrifuged at 15, 000 x g for 2 min at 4 °C. An aliquot (300 μl) of the resulting supernatant was transferred to an Eppendorf tube and treated with 60 μl of 9.3 mg/ml iodoacetic acid (pH kept in range of 8.8–9.2) and the sample was then derivatized by the addition of 300 μl of a 20 mg dansyl chloride/ml solution and incubation at room temperature overnight. After the aqueous and organic layers were separated using chloroform, materials from the top layer underwent high-performance liquid chromatography (HPLC) analysis over a Supelcosil (LC-NH₂ 25-cm × 4.6-mm, 5 μm i.d.) column in an Agilent 1200 series system (Agilent, Santa Clara, CA). Total levels of GSH were calculated relative to an internal standard (γ-glutamyl glutamate) and expressed in terms of μM GSH present. GSH levels were calculated relative to total protein content (measured by Bradford method) in corresponding samples.