Cell culture medium affects GFP photostability: a solution

penicillin, streptomycin and fetal bovine serum had no influence on EGFP photostability in cells (data not shown). We tested EGFP fused to the cytoskeletal proteins β-actin and α-tubulin and observed similar improvements in their photostabilities in DMEM – Rf and DMEM – V. Photostability of mitochondrially localized EGFP had a low dependence on medium composition and was higher than that of cytosolic EGFP in DMEM (Table 1 and Supplementary Fig. 1). We observed an enhancement of photostability for green fluorescent proteins AcGFP1 and TagGFP2 as well as for the photoactivatable fluorescent proteins PA-GFP and PS-CFP2 in their activated (green fluorescent) state (Table 1). We observed no changes in the photostability of red fluorescent proteins (DsRed2, TagRFP and mKate2) in the vitamin-depleted medium. A previous study analyzing the influence of riboflavin starvation on eukaryotic cell physiology had identified notable physiological changes occurring only after 5–7 days of culture in low-riboflavin medium3. We assessed the effect of the absence of vitamins on cell vital functions. We grew HEK293T cells in DMEM, DMEM – Rf or DMEM – V. All cells maintained normal morphology, division and attachment abilities, as well as transfection capacity over the first week. In the second week, the cells incubated with DMEM – V had depressed viability and pathologic morphology changes. HeLa cells transfected with fluorescent protein–tagged α-tubulin or β-actin had a normal cytoskeleton after 5-day culture in DMEM – V (Supplementary Fig. 2). Moreover, a standard scratch wound healing assay revealed no effect of growth in DMEM – V medium (5-day cultivation) on migration of rat embryonic fibroblasts (REF52) (Supplementary Fig. 3). We conclude that DMEM – V, and particularly DMEM – Rf, can be safely used for live-cell imaging experiments for up to several days. Our work demonstrates that the composition of the cell medium has a major effect on the photostabilities of green fluorescent proteins. Notably, photobleaching of fluorescent proteins has often been Cell culture medium affects GFP photostability: a solution