4.3. pH-Assay for Extracellular Acidification Measurements

The pH-sensitive fluorescent dye SNARF-5F (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the pH of each droplet. SNARF-5F responds to pH variations by undergoing a wavelength shift in the emission spectra. Such pH-dependent shifts allow the ratio of the fluorescence intensities from the dye at two emission wavelengths (580 nm and 630 nm) to be used for quantitative determination of pH. For each droplet the ratio of emitted fluorescence intensities at 580 and 630 nm (580/630 ratio) of SNARF-5F is calculated in real time. As the pH is more acidic, SNARF-5F fluorescence increases at 580 nm while decreases at 630 nm. To calibrate the system, Joklik’s EMEM medium titrated at different pH (7.4, 7, 6.5, 6, 5.5 and 5) was added with 4 µM SNARF-5F, emulsified and droplets screened for fluorescence. The 580/630 nm ratio were calculated for each pH and plotted against the respective pH, which allowed constructing a calibration curve.