Sodium chloride (nacl)

The pH was adjusted by adding disodium phosphate (10 mM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing sodium chloride (137 mM, FUJIFILM Wako Pure Chemical Corporation) and potassium chloride (2.68 mM, KANTO CHEMICAL, Tokyo, Japan) to potassium dihydrogen phosphate (2.0 mM) containing sodium chloride (137 mM, FUJIFILM Wako Pure Chemical Corporation) and potassium chloride (2.68 mM, FUJIFILM Wako Pure Chemical Corporation).

The strains and plasmids are listed in Table S1. All strains were derived from the K12 strain RP437, which is wild-type for chemotaxis [14 (link)]. The replacement of the wild-type cheA gene with cheA(M98L) to make CheA_S⁻ cell [15 (link)], the replacement of the wild-type fliC gene with the fliC-sticky gene [16 (link)], and the replacement of the wild-type cheZ gene with cheZ(F98S) [11 (link)] were carried out using the λ red recombinase and tetracycline sensitivity selection method [17 (link),18 (link)]. LB broth (1% bactotryptone (BD, Sparks, MD, USA), 0.5% yeast extract (BD, Sparks, MD, USA), 0.5% NaCl (Wako, Osaka, Japan)) was used for culture growth, transformations, and plasmid isolation. Tryptone broth (TB) (1% bactotryptone, 0.5% NaCl) was used to grow cells for measurements of motor rotation. Growth conditions were described in Supplemental Methods. For all measurement, the cells were suspended in 10NaMB (10 mM potassium phosphate buffer (Wako, Osaka, Japan), pH 7.0; 0.1 mM EDTA-2K (Wako, Osaka, Japan), pH 7.0; 10 mM NaCl, 75 mM KCl (Wako, Osaka, Japan)).

The preparation of hepatocytes was performed by using the collagenase perfusion method as described previously. Abdominal incisions were performed on the rats under anaesthesia, and 18G catheters (Terumo) were inserted into the PV and IVC. The initial perfusions (100 mL; flow rate, 10–15 mL/min) were performed using the perfusion solution, which included NaCl (136.89 mM, Wako), KCl (5.37 mM, Wako), NaH₂PO₄/2H₂O (0.38 mM, Wako), Na₂HPO₄/12H₂O (0.17 mM, Wako), HEPES (10 mM, DOJINDO), glucose (5 mM, Wako), ethylene glycol tetra-acetic acid (0.5 mM, DOJINDO), NaHCO₃ (4.16 mM, Wako), and phenol red (0.017 mM, Sigma-Aldrich Japan).
A secondary perfusion was performed for 8–15 min using collagenase solution (Wako), which contained NaCl (136.89 mM, Wako), KCl (5.36 mM, Wako), NaH₂PO₄/2H₂O (0.38 mM, Wako), Na₂HPO₄/12H₂O (0.17 mM, Wako), CaCl₂ (5.06 nM, Wako), HEPES (10 mM, DOJINDO), glucose (5 mM, Wako), NaHCO₃ (4.16 mM, Wako), phenol red (0.017 mM, Sigma-Aldrich Japan), and soy bean trypsin inhibitor (0.0025 mM, Sigma-Aldrich Japan). After the secondary perfusion, the liver was split into multiple tissues, and the primary hepatocytes were corrected through mesh filtration (Kawamoto, Osaka, Japan) in 2% FBS MEM.