Steriflip unit

To prepare cell-free supernatant, P. aeruginosa was inoculated from an overnight culture at an OD₆₀₀ of 0.05 and allowed to grow for 16 hours in medium with the indicated additives. The culture was centrifuged, and the supernatant was filtered with a Steriflip unit with a 0.2 μm polyethersulfone filter (MilliporeSigma). For the survival assay, S. aureus was inoculated from an overnight culture at an OD₆₀₀ of 0.05 and grown to an OD₆₀₀ of 0.5. Next, 500 μL of culture was added to 500 μL (50% v/v final concentration) of either P. aeruginosa cell-free supernatant or M63 salts control. After 16 hours, S. aureus was plated on LB agar plates and enumerated after growth.

Overnight cultures of S. aureus strains were diluted to OD₆₀₀ of 0.05 in fresh media and grown for 24 hours in flasks (or tubes or 96-well plates for NTML library supernatant preparation). Bacterial cultures grown in flasks were centrifuged at 4,000 rpm for 20 minutes. The supernatant was applied to a Steriflip unit with a 0.2 μm polyethersulfone filter (MilliporeSigma, Burlington, Massachusetts, USA). For the resuspension of S. aureus exoproducts shown in S2 Fig, 5 mL of frozen flask supernatant was lyophilized overnight and then resuspended in the same volume of either media or water. For supernatants prepared from the NTML library in 1-mL tube cultures or 96-well plates, culture was directly transferred to AcroPrep Advance 96-well 0.2 μm polyethersulfone filter plates (Pall, Port Washington, New York, USA) and centrifuged at 4,000 rpm for 10 minutes to collect the sterile-filtered flow through. Supernatants were stored at −30°C until use.

In ^Rosa26mKir6.2^NDM and ^Rosa26mKir6.2^NDM-V108E mice, the expression of mutant Kir6.2 activity occurred in the pancreas upon the excision of the Neo/WSS cassette by the tamoxifen-dependent Cre recombinase (Remedi et al., 2009 (link)). Exposure to tamoxifen permitted the translocation of Cre recombinase from the cytoplasm to the nucleus in order to perform the excision between loxP sites within the targeted Rosa26 locus. To induce the expression of mutant Kir6.2 activity and consequently the diabetic phenotype in these mutant mice (8–12 weeks of age), corn oil containing tamoxifen (75 mg/kg body weight; prepared as described below) was injected intraperitoneally for five consecutive days. Injection sites were sealed with a tissue adhesive (3M Vetbond) to prevent leakage of the injected liquid.
To make a liquid stock of tamoxifen (20 mg/ml) for injection, tamoxifen solids (Millipore-Sigma, T5648) were added into corn oil (Millipore-Sigma, C8267) in a polypropylene tube wrapped with aluminum foil to protect tamoxifen from light. The stock was placed on a nutator to dissolve overnight at room temperature. The dissolved tamoxifen stock was filtered through a sterilized Steriflip unit (Millipore) then aseptically aliquoted, stored at 4°C, and used within 5 days.