Non-invasive flow cytometric monitoring of pHi in cell culture processes using EGFP

We have described a new method for monitoring of cell culture processes using green fluorescent protein (GFP) fluorescent intensity. GFP has been used as a non-invasive fluorescent reporter for various cellular processes. In this study, enhanced (EGFP) was found to be a very sensitive indicator of pHi in in vitro cell culture, and responded rapidly to extracellular pH (pHe) changes. EGFP transfected cells were evaluated for pHi changes by flow cytometry, by measuring EGFP fluorescent intensity, and compared to that of the pH-sensitive fluoroprobe, SNARF. EGFP intensity was found to reflect pHi values of cells at different pHe in the presence of nigericin and was affected by the addition of HCl and NaOH. Significant changes in pHi were detected at different stages of batch culture and when using different cell density and media composition. The EGFP assay can be used to minimise the perturbation of cells and processes under study, thus leading to accurate information about the physiological state of single cells in a population. The results establish the application of EGFP as a non-invasive indicator of pHi for monitoring of mammalian cell culture processes.