Fig. 5 | Communications Biology

Fig. 5

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

Fig. 5

Effects of buffering regime and medium pH on intracellular pH, measured using a high-throughput imaging method. a Monolayer of DLD1 cells imaged with Cytation 5 plate reader. Image on left shows superimposition of cSNARF1 and Hoechst-33342 fluorescence maps. Image on right shows pH in individual cells, identified by nuclear staining. b Calibration curve determined from nine colorectal cancer cell lines (LS174T, PMFKO14, LS513, HCT15, SW620, GP2D, HCT116, Caco2, RW2892; seeding density 100,000 cells per well, growth area 0.56 cm2 per well). Three technical replicates each. Best-fit curve: pH = 6.978 + log((1.497 −ratio)/(ratio − 0.221)). c Histogram of intracellular pH in Caco2 monolayers bathed in D7777-based media (25 mM glucose) at pH 7.4, buffered by either 5% CO2/22 mM HCO3, or 10 mM HEPES + MES titrated to 7.4 (CO2 free). Note the substantial alkalinization in the absence of physiological buffer. Repeated three times (three technical repeats each). d Experiment performed on DLD1 monolayers. e Effect of medium pH on intracellular pH in Caco2 monolayers. The pH of CO2/HCO3-buffered media was varied by changing [HCO3] (incubation in 5% CO2). In contrast, the pH of HEPES/MES-buffered media were titrated to target pH with NaOH at the bench (incubation in 0% CO2). Best fit: linear (CO2/HCO3) or polynomial (HEPES/MES). Note that intracellular pH is more responsive to changes in extracellular pH in the absence of physiological (CO2/HCO3) buffer. f Experiment repeated on DLD1 monolayers. Data are shown as mean ± SD. Repeated three times (three technical repeats each)

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