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QUESTION: What is the stability of Hygromycin B solution?
ANSWER: Hygromycin B solution is stable for at least two years at +4°C and about one month at +37°C.

QUESTION: What color is Hygromycin B solution?
ANSWER: The color can vary from light yellow to a dark brown or caramel color. The concentrated Hygromycin solution tends to have a darker appearance.

QUESTION: What is the working concentration for selection?
ANSWER: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 25-1000 mg/ml. Commonly used concentrations for selection are 200 mg/ml for mammalian cells, 20-200 mg/ml for plant cells & bacteria cells and 200-1000 mg/ml for fungi. Your optimum concentration should be tested experimentally.

QUESTION: How can non-transfected cells escape antibiotic selection?
ANSWER: Cells can escape selection if the antibiotic is used at too low concentration or if the cell density on the plate is too high. Additionally, cells rapidly proliferating are killed faster than these, only slowly proliferating. Control cells should die within 5-7 days after addition of the antibiotic allowing colonies of resistant cells to form by 10-14 days.

QUESTION: How do I determine the Toxic Concentration?
ANSWER: Hygromycin B is added to the culture medium at a concentration that varies with the cell type transfected. A titration experiment for each cell type may therefore be performed to determine the amount of Hygromycin B needed to kill non-transfected cells. The working concentration for mammalian cell selection is normally between 50 ug/ml and 1mg/ml, Plant cells: 20-200 ug/ml, Bacteria: 20-200 ug/ml and Fungi: 200 ug-1mg/ml. Your appropriate concentration should be tested experimentally.

QUESTION: How do I perform a Dose Response curve?
ANSWER: To determine the minimum concentration of antibiotic required to kill your non-transfected host cell line. Test arrange of concentrations (5-6) to ensure that you determine the minimum concentration necessary for your cell line. Seed cells at approxiamately 20-25% confluency on the appropriate number of plates for each time plate and allow cells to adhere overnight. For cells that require higher densities for viability, increase the number of cells seeded. The next day, substitute culture medium with medium containing varying concentration of the antibiotic. Replenish the selective medium every 3-4 days. Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents the growth of non-transfected cells. Select the concentration that kills the majority of the cells in the desired number of days, usually 7-10 days.

QUESTION: How do I maintain Hygromycin resistant phenotype of transfected cell lines?
ANSWER: To maintain Hygromycin resistant phenotype of transfected cell lines and for the elimination of revertants cells may be regularly cultured in culture medium containing Hygromycin B at the same concentration used for the initial selection.

QUESTION: Replacement of Media?
ANSWER: Replacement of culture media containing Hygromycin B is needed only if nutritional components are consumed by the cells cultured. Acidification of the culture medium is a normally a sign of consumption. Utilizing phenol red or media containing phenol red will aid in the detection of acidification. In this case the media will turn yellow.

QUESTION: Is Hygromycin B sensitive to acids?
ANSWER: It is sensitive to high concentrations of acids; however, a brief exposure to dilute acids does not affect its stability.

QUESTION: Can we increase the sensitivity of our cells to Hygromycin B?
ANSWER: The sensitivity to Hygromycin B can be increased by increasing the pH of the medium. Sensitivity appears to be greater at lower salt concentrations.

QUESTION: What is the enzyme that inactivates Hygromycin B?
ANSWER: Hygromycin phosphotransferase (hpt) inactivates the antibiotic hygromycin B through phosphorylation. The hygromycin phosphotransferase gene (denoted hpt, hph or aphIV) codes for hygromycin phosphotransferase, and is utilized as selectable marker gene for both plant and animal systems.

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