Fig. 1 | Communications Biology

Fig. 1

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

Fig. 1

Measuring and setting medium pH under incubation. a Absorbance spectrum of Phenol Red (PhR) in Dulbecco’s modified Eagle’s medium (DMEM) (D7777) with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin (PS), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) plus 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and titrated (5 M HCl or 4 M NaOH) to the indicated pH. Arrows indicate wavelengths for optimal ratiometric analysis. b pH dependence of 560/430 nm ratio, fitted to curve: pH = 8.35 + log((10.9 – ratio))/(ratio – 0.0392)). c Controlling equilibrium pH by varying pCO2 and [HCO3] in DMEM (D7777) supplemented with 10% FBS and 1% PS. Dashed line plots Eq. 1. Continuous line is best fit to Eq. 3 (corrected version of Eq. 1), which accounts for buffering by serum (best fit: 1.11 mM pH−1). Inset replots the data at low [HCO3]. d Empirical determination of intrinsic buffering capacity of DMEM (D7777; 10% FBS/1% PS, 25 mM glucose) nominally lacking buffers; titration with either HCl or NaOH. Inverse of slope  provides an estimate of buffering due to serum proteins and media salts. All measurements were repeated three times (three technical replicates each). Data are shown as mean ± SEM

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