CONDITIONS OF SELECTION

Most cells growing aerobically are killed by Hygromycin B in the concentration range of 10 to 1000 µg/ml. However, the sensitivity of cells is pH dependent, i.e., the higher the pH of culture medium, the greater the sensitivity. Thus, the concentration of Hygromycin B required for complete growth inhibition of given cells can be reduced by increasing the pH of the medium. In addition, using low salt media whenever possible decreases the amount of Hygromycin B needed.

Escherichia coli :

Hygromycin-resistant transformants are selected in Low Salt LB agar medium (yeast extract 5g/l, tryptone 10 g/l, NaCl 5g/l, agar 15 g/l, pH 8) supplemented with 100 µg/ml of Hygromycin B. Plates containing Hygromycin B are stable for 1 month when stored at 4°C. For optimum results the use of Fast-Media® HYGRO is recommended.

Other bacteria :

Working concentration for most bacteria is within the 20-200 µg/ml range.

Mammalian cells :

The working concentration of Hygromycin B for mammalian cell lines varies from 50 to 1000 µg/ml. It is recommended that an initial experiment be done to determine the optimal concentration of Hygromycin B required to kill your host cell line. The killing and the detachment of dead cells from the plate, specially at high cell density, can require a longer time compared to G418. Foci of Hygromycin-resistant stable transfectants are usually individualized after 10 days to 3 weeks incubation, depending on the cell line. Suggested concentrations of Hygromycin B for selection in mammalian cells are listed below.

Cell line
Species
Tissue
Culture medium
Hygromycin (µg/ml)
293
Human
Kidney
DMEM
100
HeLa
Human
Uterus
DMEM
200
B16
Mouse
Melanoma
RPMI
50-100
PC1.0
Hamster
Pancreatic adenocarcinoma
RPMI
100-200
C6
Rat
Glial cells
DMEM
100-200

Plant cells :

Working concentration for plant cells is usually within the 20-200 µg/ml range.

Yeasts :

Use 50 µg/ml to 200 µg/ml.

Fungi :

Use 100 µg/ml to 300 µg/ml. For efficient selection, it is best to work in media with low salt concentration and pH 6 to 7.

 

References

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