Substitution of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) for bicarbonate in protein-free animal cell culture medium: application to vaccinia virus quantitation and fluorogenic acetylesterase assay in living LM cells

Publisher Website
Google Scholar
Abstract
The buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at 15 mM has been substituted for sodium bicarbonate in the protein-free medium used for the growth of LM mouse fibroblasts. Cells have been cultivated in spinner flasks for up to 6 months with continuous aeration from an air line. The generation time was similar to that of parallel bicarbonate-buffered cells. Cotton-plugged shaken Erlenmeyer flasks produced the same results. HEPES-buffered cells have been grown in cotton-plugged prescription bottles and in glass and plastic Petri dishes in an ordinary humidified incubator. Vaccinia virus plaque production was slightly reduced in cells grown and overlaid with HEPES medium compared to bicarbonate medium. There was little difference in plaque count on cells grown in bicarbonate and overlaid with HEPES medium. These results indicate that HEPES buffer could be used routinely for cell and virus growth in aerated systems where CO2 is not limiting. Acetylesterase (EC. 3.1.1.6), measured by the fluorometric breakdown of fluorescein diacetate by living cells, was lower in HEPES than in bicarbonate-buffered medium, indicating an effect of HEPES on the cell membrane. The ability of HEPES to withstand autoclaving also renders it useful as a buffer for currently available autoclavable Eagle's medium.

This publication has 0 references indexed in Scilit: