Quant it picogreen dsdna reagent

Double-stranded DNA was measured in the BALF using Quant-iTPicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. For dsDNA measurement using Quant-iTPicoGreen dsDNA reagent, cells were stimulated in a DMEM high glucose without phenol red medium (ThermoFisher Scientific, Waltham, USA).

mtDNA isolation and damage measurement were performed as previously described by Barchiesi et al. [42 (link)]. Briefly, mtDNA was extracted by patients isolated mitochondria from non-tumor or HCC sample using a plasmid isolation kit [43 (link)] and quantified by Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen). Q-PCR was performed on each sample to amplify a 16 ∼ Kbp fragment, using the following primers: FOR 5′-TCT AAG CCT CCT TAT TCG AGC CGA-3′ and REV 5′- CCA TCC AAC ATC TCC GCA TGA TGA AA-3′. Fluorescence readings of the Q-PCR reactions were quantified in triplicate with Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen) and then averaged for each sample. Blank value was subtracted and the ratio of the fluorescence readings obtained for the tumor tissue to those of the distal section determined the relative amplification of the mtDNA for each patient sample. Relative mtDNA damage was then expressed as the inverse of this relative amplification.

A Glutamine/Glutamate Determination Kit (Sigma Aldrich) was used to measure the decrease of glutamate in the cell culture media over time. Assays were initiated 72 h after plating in order to let the cells recover. Cells were seeded at 70,000 cells/cm². Before the assay cells were washed with HBSS (Invitrogen) buffer and incubated with HBSS (Invitrogen) for 30 min. To evaluate the contribution of EAAT1, 100 µM l-glutamic acid (Invitrogen) was prepared with the EAAT1 inhibitor UCPH 101 (Abcam, Cambridge, UK) at 1.34 µM, the solution was prepared in HBSS (Invitrogen) with or without inhibitor and incubated with the cells. After 60 min the remaining glutamate concentration in the media was measured following enzymatic reaction at 340 nm using a Multi-label reader (Perkin Elmer, Waltham, MA, USA). After subtraction of background (blank sample containing 0 nM glutamate), the decrease of glutamate in the media was determined using the glutamate standard prepared according to manufacturer’s instructions. Directly after sample isolation, analysis of double stranded DNA content in each well was performed with Quant-iT™ PicoGreen™ dsDNA Reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The double stranded DNA content was used to normalize glutamate uptake data.