Bp338 500

The Erdman strain was used for all M. tuberculosis (Mtb) infections as well as a luciferase expressing strain. Low passage lab stocks were thawed for each experiment to ensure virulence was preserved. Mtb was cultured in roller bottles at 37°C in Middlebrook 7H9 broth (BD Biosciences, GTIN00382902713104) supplemented with 10% OADC (BD Biosciences, 212351), 0.5% glycerol (Fisher, G33-1), and 0.1% Tween-80 (Fisher, BP338-500). All work with Mtb was performed under Biosafety level 3 containment using procedures approved by the Texas A&M University Institutional Biosafety Committee.

Alzet® Mini-Osmotic Pumps (model 2004) were loaded with FMF-04-159-216 (R&D Systems 7158, Minneapolis, MN, USA) 1.47 μg/uL in vehicle solution containing 8% DMSO (Fisher Scientific, BP231, Hampton, NH, USA), 2% Tween 80 (Fisher Scientific, BP338-500) and 90% ddH₂O) 16 h before stereotactic surgeries. Brain infusion catheters with 3.5 cm long catheter tubing (Alzet® Brain Infusion Kit) were attached as per manufacturer’s instructions. Brain infusion assemblies were incubated at 37 °C in sterile saline until implantation. 4-month-old PAC α-Syn^A53TTG were deeply anesthetized with isoflurane for the stereotactic implantation of brain infusion assemblies. FMF-04-159-2 (release rate of 0.35 mg/kg/day) or its vehicle solution was continuously administered into the cerebral ventricles (coordinates relative to bregma: −1.1 mm medial-lateral; −0.5 mm antero-posterior and −3 mm dorso-ventral) for 28 days with the brain infusion catheter attached to the skull and the connected pump in a subcutaneous pocket of the mouse’s back. The body weight of mice was measured and their activity, neurological signs, facial grimace, coat condition, and respiration were scored (from 0 to 3) within 28 days after the surgery. Mice were sacrificed and organs were collected on the 28th day of the administration period. 10.17504/protocols.io.x54v9pqy1g3e/v1.

The PKTP buffer was composed of 0.1% Tween 80 (BP338-500 Fisher Scientific, Waltham, MA, USA), 5 mg/mL Poly Vinyl Sulfonic Acid sodium salt solution (278416 Millipore Sigma, St. Luis, MI, USA), and 200 µg/mL Proteinase K (Zymo Research, Irvin, CA, USA) in Ca- and Mg-free Dulbecco’s Phosphate-Buffered Saline buffer (DPBS L0615-500 Biowest, Riverside, MO, USA). The saliva samples were mixed with PKTP on a 1:1, 2:1, or 3:1 saliva:PKTP ratio and was heat inactivated (10 min at 96 °C) on a SimpliAmp Thermal cycler (Applied Biosystems, Waltham, MA, USA). Then, 4 µL of the inactivated saliva:PKTP mix was used for single- or multiplex RT-PCR reactions. The PKTP buffer was freshly prepared for all experiments except for the stability tests.