Nahco3

MH broth (Oxoid Ltd., Brno, Czech Republic) supplemented with NaHCO₃ was prepared as follows: after sterilization (121 °C, 15 min), the media were cooled down to 4 °C, and NaHCO₃ (50 mmol/L; Sigma-Aldrich Co., Prague, Czech Republic), and meropenem (1 mg/L; AstraZeneca UK Ltd., Macclesfield, UK) were added.

Liver-resident immune cells were isolated as previously described (21 (link)). Briefly, 100 ml of warmed 1× Hank’s balanced salt solution (HBSS, Worthington, Lakewood, NJ, USA) containing 25 mM N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid (HEPES) (Sigma-Aldrich), 4.2 mM NaHCO₃ (Sigma-Aldrich), and 83 μM Ethylenediaminetetraacetic acid (EDTA) (Sigma- Aldrich) was perfused through the portal vein. Subsequently, 100 ml of warm 1× HBSS with 25 mM HEPES, 4.2 mM NaHCO₃, 1.26 mM CaCl₂ (Sigma-Aldrich), 490 μM MgCl₂ (Sigma-Aldrich), 406 μM MgSO₄ (Sigma-Aldrich), and 50 mg of Collagenase Type IV (Worthington) was perfused through the liver for 15 min to disrupt the extracellular matrix. Tissue was then mechanically disrupted and passed through a 100-μM cell strainer. Hepatocytes were pelleted using centrifugation at 70g for 3 min, and non-parenchymal cells (NPCs) in the supernatant were transferred to a fresh tube. After a second hepatocyte-removal step, the NPCs were pelleted at 300g centrifugation for 5 min. RBCs were lysed by incubation in 2 ml of ACK buffer (Sigma-Aldrich) for 2 min at room temperature. NPCs underwent a 25:50 Percoll gradient to isolate liver-resident immune cells for downstream analyses.

Room-temperature burst kinetics were obtained under burst-phase conditions using an SX20 stopped flow (Applied Photophysics) by monitoring substrate depletion by absorbance at 260 nm. Enzyme and substrate were mixed 1:1 at 25 °C and in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2) supplemented with 50 mM NaHCO₃ (Sigma-Aldrich). Burst kinetics were assayed at final enzyme concentrations of 10 μM and final substrate concentrations varying between 50 and 400 μM (equation (1)). $\frac{P}{E_0}=v_{\mathrm{steady}}\times t-\left(v_{\mathrm{steady}}-v_{\mathrm{burst}}\right)\times \left(1-e^{-k\times t}\right)/k$
Catalytic parameters (k_cat, K_M and k_cat/K_M) were determined under burst-phase and steady-state conditions using CAZ (Δξ = −9,000 M⁻¹ cm⁻¹) at 260 nm by measuring the initial enzymatic reaction rate in an Epoch plate-reader (Biotek). Burst phase rates were determined at 4 °C, and steady-state parameters were determined at 25 °C. Reactions rates were obtained in at least duplicates at a final enzyme concentration of 1 μM (final assay volume of 100 μl). Ultraviolet-transparent 96-well plates (Corning) were used. Assays were performed in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2), supplemented with 50 mM NaHCO₃ (Sigma-Aldrich).

Mice were killed 3 h following water or saccharin consumption by decapitation, following anesthesia with isoflurane. Brain slices were cut at 300 μm in coronal sections using a vibratome (Campden-1000) in ice chilled cutting solution (110 mm sucrose, 60 mm NaCl, 3 mm KCl, 1.25 mm NaH₂PO₄, 28 mm NaHCO₃, 0.5 mm CaCl₂, 7 mm MgCl₂, 5 mm D-glucose, and 0.6 mm ascorbate, Sigma-Aldrich). The slices were placed in artificial CSF (ACSF; 125 mm NaCl, 2.5 mm KCl, 1.25 mm NaH₂PO₄, 25 mm NaHCO₃, 25 mm D-glucose, 2 mm CaCl₂, and 1 mm MgCl₂, Sigma-Aldrich) for a 30 min recovery period at 37°C and then kept at room temperature for at least an additional 30 min before electrophysiological recording. Throughout, ACSF was continually gassed, using carbogen (O₂ 95%, CO₂ 5%).

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental cultures was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentrations: chloramphenicol, 10 μg mL⁻¹; spectinomycin, 25 μg mL⁻¹; erythromycin, 25 μg mL⁻¹; and kanamycin, 25 μg mL⁻¹). All experimental cultures were prepared in quadruplicates. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliters of culture were sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The pH of experimental cultures was measured with MColorpHast™ pH-indicator strips (pH 6.5–10) (Merck) and the cultures pH were adjusted to the range between 7–8 using 37% HCl (Sigma-Aldrich).

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental culture was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentration: chloramphenicol 20 μg mL⁻¹, spectinomycin 50 μg mL⁻¹, and kanamycin 50 μg mL⁻¹). All experimental cultures were prepared in triplicate. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliter of culture was sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The cultivation was terminated when the isobutanol production in the culture started to decrease.