Antibleaching live cell visualization medium
Photobleaching of fluorescent proteins in response to prolonged exposure to exciting radiation significantly impacts their performance as in vivo labels. It has been shown that oxidative reddening of green fluorescent proteins is one of the main sources of GFP photobleaching (Bogdanov et al., 2009). Evrogen's new DMEMgfp-2 is a minimally depleted live cell visualization medium that excludes the components responsible for this effect (Bogdanov et al., 2012). DMEMgfp-2 is an improvement over DMEMgfp (referred to as DMEM-V in Bogdanov et al., 2009), exhibiting similar green fluorescent protein photostability without depleting as many of the compounds normally present in most common cell culture media. This modification makes DMEMgfp-2 better suitable for long-term experiments.
Replacing the culture medium with DMEMgfp-2 for the period of visualization results in up to a 9-fold increase of photostability of EGFP, a 3.3-fold increase of photostability of TagGFP2 and more than a 4-fold increase of photostability of activated forms of photoactivatable PA-GFP and PS-CFP2 (the effect varies for particular fusions and localizations).
Rutin (plant flavonoid glycoside also known as vitamin P) is the component that inhibits oxidative reddening and thus increases EGFP photostability in the cell culture experiments (Bogdanov et al., 2012). An increase of EGFP photostability comparable to the level observed in DMEMgfp-2 is achieved when rutin is added 30 min before imaging to standard DMEM. Addition of rutin to DMEMgfp-2 results in further enhanced EGFP photostability (approximately 1.5-fold).
30 min before starting imaging experiment replace the cell culture medium with DMEMgfp-2 as is or supplemented with rutin at a final concentration of 20 mg/l. Always prepare a fresh solution of rutin in DMEMgfp-2! The replacement should be performed under sterile conditions.
For long experiments DMEMgfp-2 can be supplemented with L-glutamine, penicillin, streptomycin and fetal bovine serum.
Long-term fluorescent microscopy of EGFP in live HEK293T cells maintained in DMEM or DMEMgfp.
Influence of cell medium on photostability of fluorescent proteins.
(A) – EGFP, (B) AcGFP1, (C) TagGFP2. Graph shows normalized bleaching curves of fluorescent proteins in live HEK293 cells maintained in DMEM (black lines), or DMEMgfp (green lines). Standard deviations (n = 15-20 cells) are shown.
HeLa cells transfected with fluorescent protein-tagged α-tubulin or β-actin had a normal cytoskeleton after 5-day culture in DMEMgfp.
Fluorescence microscopy of TagGFP2-
Influence of rutin on EGFP photobehavior.
Bleaching of green fluorescence in EGFP-expressing live HEK293 cells maintained in DMEM (red), DMEM with rutin (blue), DMEMgfp-2 (violet), or DMEMgfp-2 with rutin (green). Green fluorescence intensities in individual cells are background subtracted and normalized to maximum (initial) value in each cell. Standard deviation values (n = 15–20 cells in a representative experiment out of five independent experiments) are shown.
Data from Bogdanov et al., 2012.
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|MC102||antibleaching live cell visualization medium|
Rutin, 100x solution
Shipping: ambient temperature.
Storage: DMEMgfp-2 medium should be stored at +4 – +8°C. Rutin is stored at -20°C.
Upon receipt place rutin in the freezer. DO NOT FREEZE DMEMgfp-2.
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