C di amp

To allow for cytosolic access, culture filtrates, molecules or enzymes were diluted in Opti-MEM® and added to macrophages with a final concentration of 10 µg/ml digitonin (Sigma). Cells were incubated for 30 min at 37 °C in 5% CO₂ before diluting out the permeabilizing effect of digitonin by addition of x10 volume of pre-warmed Opti-MEM®. After an additional incubation of 20 min at 37 °C in 5% CO₂, all medium was removed and replaced with fresh Opti-MEM®. RNA was isolated for RTqPCR analysis at 2.5 h post treatment. Molecules and enzymes were added at the following final concentrations unless otherwise indicated: Purified recombinant NADase or NADaseG330D (G330D) 0.5 µM^{34 (link)}, c-di-AMP 7.5 µM (InvivoGen), c-di-GMP 7.5 µM (InvivoGen), dsDNA/pTEC15 2.5 ng/ml (Addgene), pI:C 2.5 µg/ml (InvivoGen), LPS 2.5 µg/ml (Sigma). ATP (Thermo Fisher Scientific) was used at final concentrations of 0.1 mM or 1 mM. To degrade c-di-AMP, samples were preincubated (at 32 °C for 60 min) with phosphodiesterase I (Sigma) at a final concentration of 20 U/ml.

Using aseptic technique, femurs were harvested, and the bone marrow flushed from C57BL/6 and Tmem173^gt mice for macrophage polarization studies. The BMDMs were expanded in culture for 7 d in the presence of L-929 mouse fibroblast conditioned medium as a source of M-CSF. Cells were re-seeded onto 6-well plates at 2 × 10⁶ cells/well and polarized for 48 hrs with the addition of 50 ng/ml LPS (List Biological Labs) and 50 ng/ml murine IFNγ (Cedarlane) for M1, 40 ng/ml murine IL-4 (R&D Systems) for M2 or left untreated for M0 cells. These cells were subsequently treated with either 20 μg/ml DMXAA (Cedarlane), 20 or 40 μg/ml 2′3′-cGAMP or 20 μg/ml c-di-AMP (InvivoGen, San Diego, CA) for an additional 6 hrs in the absence of polarizing cytokines. cDN treatments were given in the presence of Lipofectamine-2000 (LF2000, Invitrogen) to allow for entry across the cell membrane¹¹ . BMDM cultures were all maintained at 37 °C in a 5% CO₂ humidified incubator.