Butyrate, succinate, lactate, and acetate concentrations in culture supernatants were quantified as described previously (Clark et al., 2021 (link)). Supernatant samples were thawed in a room temperature water bath before addition of 2µL of to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 2400xg for 10 min and then 150µL of each sample was filtered through a 0.2µm filter using a vacuum manifold before transferring 70µL of each sample to an HPLC vial. HPLC analysis was performed using a Shimadzu HPLC system equipped with a SPD-20AV UV detector (210 nm). Compounds were separated on a 250×4.6 mm Rezex OA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 ml min-1 and at a column temperature of -50°C. The samples were held at 4°C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N ( ). At least two standard sets were run along with each sample set. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. The injection volume for both sample and standard was 25µL. The resultant data was analyzed using the Shimadzu LabSolutions software package.
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