Quantitative PCR

N9 microglial cells are treated either with 1 mM DMAG (Sigma-Aldrich Cat. No. D4268) alone or pre-incubated with NEM 1 µM for 15 min before addition of 1 mM DMAG for 6 h. Total RNA was extracted using HiPur A Total RNA Mini prep Purification kit (Himedia Cat. No. MB 602). 1 µg of mRNA was prepared for cDNA synthesis using cDNA synthesis kit (BIORAD, iscript cDNA synthesis kit Cat. No. 1708891). After cDNA preparation, quantitative polymerase chain reaction (Q-PCR) was performed in triplicates and two biological replicates (BIORAD iTaq Universal SYBR green qPCR super mix, Cat. No. 1725121) using QuantStudio 5 (Thermo scientific). Primer blast, an online tool developed at NCBI was used to design the primer sequences encoding for HPRT (hypoxanthine guanine phosphoribosyl transferase), TNFα, IFNy, TGFβ, IDO-1, IDO-2, TDO-2 and P2 receptors (P2X₁, P2X₂, P2X₄, P2X₅, P2X₇, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁). The primer sequences used in this study are provided in Supplementary Table ^S5. Expression of target genes were normalized to expression of HPRT. The difference between samples and controls were calculated using Delta-delta C_t method. In case of NEM (N-Ethylmaleimide, Sigma-Aldrich, Cat. No. E3876), Sodium Nitroprusside (Sigma-Aldrich, Cat. No. 71778), PPADS (abcam, Cat No. ab120009), P2X₇ inhibitor (A-438079 HCl) (Cat. No. S7705) and BAPTA-AM, cell permeant chelator (Thermo Fischer Scientific, Invitrogen, Cat. No. B1205) experiments, N9 microglial cells were either treated with vehicle, 1 mM DMAG or pre-treated with NEM (1 μM) or PPADS (10 μM) or P2X₇i (10 μM) 15 min before addition of 1 mM DMAG and subsequently incubated for 6 h. N9 cells were treated with BAPTA-AM in RPMI medium without serum for 30 min. After 30mins of incubation, the medium was changed to complete RPMI medium followed by the addition of DMAG or ATP (25 µM or 100 µM), with or without BAPTA and incubated for 6 h. The gene expression analysis of cytokines (TGFβ, TNFα and IFNɣ) as well as IDO-1, IDO-2 and TDO-2 were carried out using Q-PCR.