Eukaryotic Cell Culture Applications
Hygromycin B is routinely used as a selective agent in mammalian cell culture to isolate Hygromycin B resistant cells after transfection. Selectable markers for Hygromycin B resistant cells include the hyg or hph resistance genes which express a phosphotransferase that inactivates Hygromycin B by phosphorylation. Effective working concentrations range from 100 – 1000 µg/mL. The optimal working concentration of hygromycin B for selection of resistant mammalian clones depends on the cell lines used, hygromycin B quality, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the hphresistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of Hygromycin B. Optimal selection concentrations of Hygromycin B can range from 50 µg/mL - 1000 µg/mL; however, most common selection concentration ranges are between 50 µg/mL - 200 µg/mL.
For additional information regarding relevant cell lines, resistance plasmids, and culture media, please visit our Cell Culture Database.
Microbiology Applications
Hygromycin B can be used as a selection agent to isolate Hygromycin B resistant bacteria and fungi. The following Hygromycin B selection concentrations should serve as a guide only and may vary depending on experimental conditions and cells used:
- Bacteria (E. coli) - 50 µg/mL - 100 µg/mL
- Fungi - 100 µg/mL - 300 µg/mL
- Yeasts - 50 µg/mL - 200 µg/mL
Plant Biology Applications
Hygromycin B is routinely used as a selection agent for Arabidopsis plants that have been transformed with a hygromycin B resistance gene. A rapid method to screen for hygromycin B resistant Arabidopsis in less than four days has been developed. After Arabidopsis seeds have been transformed with a resistance plasmid (pBIG-HYG), they are plated on MS medium with hygromycin B and subjected to a two day stratification at 4°C in the dark. Seeds are then exposed to light for 4-6 hours to stimulate germination and then placed in the dark for another two days. Transformed seeds are selected and identified after a 24 hour period in the light. Resistant transformants are characterized by long hypocotyls. (Harrison et al, 2006).