Rpmi 1640 without glucose

To select and maturate cardiomyocytes, the differentiated cells were cultured for 10 days under one of two conditions: RPMI1640 without glucose (Life Technologies, U.S.A) supplemented with 4 mM sodium DL-lactate (Sigma-Aldrich, U.S.A) and B27-insulin (GIBCO, U.S.A); or RPMI1640 without glucose (Life Technologies, U.S.A) supplemented with 5 mM sodium DL-lactate (Sigma-Aldrich, U.S.A), 500 µg/mL human albumin (Sigma-Aldrich, U.S.A), and 211 µg/mL L-ascorbic acid (Sigma-Aldrich, U.S.A). The medium was replaced every other day.

The Helicobacter pylori strain 67:21 has previously been described^{50 (link)}. The H. pylori mutants 67:21ΔarsS, 67:21ΔcheY and 67:21ΔtlpC were designed and constructed in this study. All H. pylori were grown on Columbia blood agar plates (Thermo Fisher) supplemented with 8% defibrinated horse blood and 8% inactivated horse serum (Håtunalab) for 3 days at 37 °C under microaerophilic conditions, i.e., in an incubator with 5% O₂, 10% CO₂ and 85% N₂. Before each experiment, bacteria from the agar plates were suspended in Brucella broth (Acumedia) containing 8% heat-inactivated fetal bovine serum (FBS) (Sigma Aldrich) and cultured for 24 h under shaking and microaerophilic conditions. At the time of experiment the medium was RPMI 1640 without glucose (Thermo Fisher). Escherichia coli strain DH5α (Invitrogen) was used in the construction of 67:21ΔarsS, 67:21ΔcheY and 67:21ΔtlpC and was cultured in LB medium at 37 °C. When the bacteria were required to be cultured on selective media, chloramphenicol was supplemented at the following concentrations: 5 μg/ml added to CBA plates for H. pylori and 10 μg/ml added to LB plates for E. coli.H. pylori strain 26695 (ATCC 700392) and J99 (ATCC 700824) have previously been described and was used to verify results as compared to strain 67:21.