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Prior to exposure to LPS (Sigma Cat. no L5024, E. coli O127:B8) at a concentration of 100 ng/ul, cells were pretreated for 1 h with either 10 nM AR-R17779 hydrochloride (Tocris Bioscience Cat# 3964), a selective α7nAChR agonist, or 100 nM α-Bungarotoxin (Tocris Bioscience Cat# 2133), a selective α7nAChR antagonist. Optimal dose of AR-R17779 (A) or α-Bungarotoxin (B) was chosen based on a dose-response experiment with LPS. We have tested 0, 10, 100, and 1,000 nM of α-Bungarotoxin and 0, 1, 10, and 100 nM of AR-R17779 in the absence or presence of 100 ng/ul LPS, and measured IL-1β concentrations in cultured media as the endpoint. The 100 nM α-Bungarotoxin and 10 nM AR-R17779 were chosen because the cells responded in a linear range as indicated by IL-1β production.
AR-R17779 was reconstituted in DMSO as stock solution, serial dilutions were made to prepare the working stock; to obtain 10 nM AR-R17779 in concentration per well, 5 ul working stock was added well by well containing 500 ul media; only DMSO was added in control well, therefore, the DMSO concentration per well was 1%. α-Bungarotoxin was reconstituted with culture media into a stock solution and underwent serial dilutions.
In a complete cell culture experiment, we had four experimental groups: Control (naïve control), LPS100 (naïve LPS), LPS100+B100 (naïve LPS+B) and LPS100+A10 (naïve LPS+A). Second hit cell cultures were designed with the same pattern and divided into four experimental groups: Control (SHC), LPS100, LPS100+B100 and LPS100+A10.
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