Ppads tetrasodium salt

EpiLife culture medium with Ca²⁺ (60 μM), EpiLife defined growth supplement (EDGS), penicillin/streptomycin, and Lipofectamine 2000 reagent were purchased from Thermo Fisher Scientific (Waltham, MA USA). On-target plus siRNA was purchased from GE healthcare (Little Chalfont, UK). FNC coating mixture was purchased from Athena ES (Baltimore, MA, USA). Suramin hexasodium salt (an inhibitor of P2X and P2Y receptors), PPADS tetrasodium salt (an inhibitor of P2X and P2Y receptors), TNP-ATP triethylammonium salt (an inhibitor of P2X receptor), NF 157 (an inhibitor of P2Y₁₁ and P2X₁), ARL 67156 (an inhibitor of ecto-ATPases), and ¹⁰Panx (an inhibitor of Pannexin-1 channel) were purchased from Tocris Bioscience (Minneapolis, MN, USA). Adenosine-5’-(γ-thio)-triphosphate tetralithium salt (ATPγS, a non-hydrolyzable ATP analogue), Brilliant blue G (BBG; an inhibitor of P2X receptors), and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, USA).

To block voltage-gated sodium channels, 1 μM Tetrodotoxin (Cat#1078; Tocris) was applied to the media before electrostimulation of spheroids. Control cells were treated with DMSO as vehicle. The following drugs and final concentrations were applied for pharmacological blockade on iMGLs exposed to spheroid conditioned media: 100 μM AR-C 66096 Tetrasodium Salt (Cat#3321; Tocris), 200 μM (+)-MK 801 Maleate (Cat#0924; Tocris), 200 μM PPADS Tetrasodium Salt (Cat#0625; Tocris), 200 μM MPEP Hydrochloride (Cat#1212; Tocris), 50 μM L-AP4 (Cat#0103; Tocris), 10 μM YM 58483 (BTP2) (Cat#3939; Tocris), 10 μM CNQX Disodium Salt (Cat#1045; Tocris), 50 μM D-AP5 (APV) (Cat#0106; Tocris), 100 μM PTX (Picrotoxin) (Cat#1128; Tocris). To induce lipid accumulation, 20 μM Oleic Acid (Cat# 03008; Sigma-Aldrich) was applied overnight to iMGLs, control cells were treated with 0.1% BSA as vehicle. 1 μM Triacsin C (Cat#10007448; Cayman Chemical) was applied to iMGLs overnight to deplete lipid accumulation.