Synthetic mucus was prepared similar to Song et al. Briefly, reconstituted from porcine gastric mucus (PGM, CAS M2378) in P/S-free complete media (4% w/v), left to stir for 2 h (room temperature, 100 r.p.m.). 4-Arm PEGthiol (PEG-4SH, Laysan Bio Inc., 174-47), a cross-linking agent, was prepared in P/S-free complete media (4% w/v), and mixed at equal volumes with the PGM solution, resulting in a final 2% w/v of both PGM and PEG-4SH. (S1) δH2 is the limiting chemical shift of the indicator in its doubly protonated state, however this second protonation step (pKa2) cannot be accessed with our pH gradient. Nevertheless, only the first protonation step (pKa1) need be considered in our experiments as pH>4 in all cases. Finally, the 'mixed' pKa in the media used for cell culture (ionic strength, I = 0.174 M) was calculated from the 'mixed' pKa (pKa*), measured as described in the paragraph above and as obtained via fitting to Equation S1, by first correcting it to the thermodynamic pKa (pKa,0) using Equation S2. p𝐾 a,0 = p𝐾 a