Cell culture medim, vitamins and fluorescent proteins photostability

Cell culture medium affects GFP photostability: a solution

Alexey M Bogdanov 1, Ekaterina A Bogdanova 1, Dmitriy M Chudakov 1, Tatiana V Gorodnicheva 2, Sergey Lukyanov 1 & Konstantin A Lukyanov 1

1 Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
2 Evrogen JSC, Moscow, Russia.
e-mail: kluk@ibch.ru

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 measured in PBS 4, which may result in an overestimation of photostability compared to commonly used live-cell imaging conditions. The use of media depleted of vitamins for fluorescence imaging of live cultured cells appears to be a simple and efficient way to improve the performance of some widely used fluorescent proteins in various ensemble and single-molecule applications 1,5,6.

Minimum Essential Medium Eagle

Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met by Eagle's Basal Medium (BME). Subsequent studies using these and other cells in culture indicated that additions to BME could be made to aid growth of a wider variety of fastidious cells. MEM, which incorporates these modifications, includes higher concentrations of amino acids so that the medium more closely approximates the protein composition of mammalian cells. MEM has been used for cultivation of a wide variety of cells grown in mono-layers. Optional supplementation of non-essential amino acids to the formulations that incorporate either Hanks' or Eagles' salts has broadened the usefulness of this medium. The formulation has been further modified by optional elimination of calcium to permit the growth of cells in suspension.

Most of the formulations offered contain the original Earle's salts. However, a number of formulations contain Hanks' salts. Both of these phosphate and bicarbonate buffered saline solutions contain the same ions: calcium; magnesium; potassium; sodium,and phosphate. They differ in the concentrations of calcium, sodium and in the buffer component. Hanks' buffered salts solution contains a substantially lower concentration of sodium bicarbonate than does Earle's buffered salts.

The original MEM formulation contained Earle's salts and a group of amino acids generally referred to as essential amino acids. They were considered essential because the cells for which the medium was developed would not grow in their absence. The 12 essential amino acids are: L-arginine; L-cystine; L-glutamine; L-histidine; L-isoleucine; L-leucine; L-methionine; L-phenylalanine; L-threonine; L-tryptophan; L-tyrosine; and L-valine.

* not all MEM has 2.2 gm/L sodium bicarbonate; all MEM should have 1 gm/L glucose