Fig. 4 | Communications Biology

Fig. 4

From: Evidence-based guidelines for controlling pH in mammalian live-cell culture systems

Fig. 4

Enhancing buffering capacity of CO2/HCO3-containing media with non-volatile buffers, with consideration of the CO2-HCO3 equilibrium. a Medium (DMEMD 7777, 10% FBS, 1% PS, 25 mM glucose) supplemented with non-volatile buffer HEPES and MES (10 mM), and titrated to indicated target pH (large circles). NaHCO3 then added to a concentration expected to be in equilibrium with 5% CO2 at target pH (Fig. 1c). Time course of pH equilibration under 5% CO2 from different starting levels. Repeated  three times (three technical replicates each). b Good agreement between target and measured equilibrium pH. c Time course of medium acidification in Caco2 cells (seeding density 4,000 cells per well, growth area of 0.32 cm2 per well). Media buffered with 5% CO2/22 mM HCO3, or the combination of CO2/HCO3 plus 10 mM HEPES/MES (a 19% increase in time-averaged buffering, β). Medium lactate accumulation at the end point was greater with enhanced buffering. d Experiment performed on DLD1 cells. e Experiment performed on DLD1 cells with 30 mM HEPES/MES. f Experiment repeated from a more acidic starting pH, at which 30 mM HEPES/MES is expected to provide half of total buffering. Measurements repeated four times (three technical replicates each). Statistical tests: lactate measurements tested by two-sided t test (*P < 0.05, **P < 0.01); time courses tested by two-way analysis of variance (ANOVA) (P value for the effect of buffering is stated). g Increasing buffering capacity with non-volatile buffers also increases osmolarity due to the buffer molecules and the base required for titration. Calculated [NaOH] required to titrate MES, PIPES or HEPES buffer to a target pH. h Osmolality of three different media formulations. Arrows show gap in osmolality in HCO3-free media, which can be filled with buffer and acid–base required for titration, plus additional NaCl required to bring osmolality to a physiological level. Note, in the case of medium D7777 and D1152, a total of 88 and 132 mOsm kg−1 can be added, respectively. i Free [Ca2+] measured by electrode, showing partial Ca2+ chelation by non-volatile buffers. Data are shown as mean ± SEM. Repeated three times

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