Instructions

1. Preparation of storage solution (50 mg/mL) 

Weigh 0.5 g hygromycin B and add 10 mL 1×PBS, PH 7.4. After fully dissolved, filtrate with a 0.22 μm filter for sterilization. Aliquot the sterilized solution and store at -20℃.

2. Commonly used screening concentration

The working concentration of hygromycin B for screening stable strains varies according to cell type, culture medium, growth conditions and cell metabolic rate. It is recommended to establish a kill curve (dose-response curve), to determine the optimal screening concentration for the first time. 

Generally, mammalian cells: 50-500 μg/mL; Bacterial/plant cells: 20-200 μg/mL; Fungi: 300-1000 μg/mL.

3. Establishment of the killing curve

[Note]: In order to screen stable cell lines, it is necessary to determine the minimum concentration of antibiotics capable of killing untransfected host cells. This can be achieved by establishing a killing curve (dose-response curve). At least five concentrations should be arranged.

1) Day 1: Untransformed cells are planked in an appropriate culture plate at a cell density of 20-25% and cultured overnight. 

[Note]: The numble of inoculation cells can be increased for cells requiring higher density to detect vitality. 

2) Set the concentration gradient within the appropriate range according to the cell type. Mammalian cell can set 50, 100, 250, 500, 750, 1000 μg/mL. Dilute the Hygromycin B solution 1:10 with deionized water or PBS buffer to 5 mg/mL. And then dilute the solution to the corresponding working concentration according to the following table.

Final Concentration (μg/mL)	Medium Volume (mL)	Addition Volume of 5 mg/mL Hygromycin B (mL)
50	9.9	0.1
100	9.8	0.2
250	9.5	0.5
500	9.0	1.0
750	8.5	1.5
1000	8.0	2.0

3) Day 2: Replace with a freshly prepared medium containing the corresponding concentration of the drug. Make three parallel samples for each concentration. 

4) Replace with fresh media containing drugs every 3-4 days. 

5) Living cell counts are performed at a fixed cycle (e.g., every 2 days) to determine the appropriate concentration to prevent the growth of untransfected cells. Choose the minimum concentration which kills the majority of cells within an ideal number of days (usually 7-10 days), as the working concentration for screening stable cell line. 

4. Screening of stable transfected cells

1) 48 h after transfection, the cells were subcultured by screening medium containing hygromycin B at an appropriate concentration (direct or diluted). 

[Note]: Antibiotics work best on actively dividing cells. If the cells are too dense, the antibiotic will not kill the cells. Split the cells such that the cells are no more than 25% confluent. 

2) Change the screening medium every 3-4 days. 

3) Measure the cell colony formation after 7 days of screening. Colony formation may take another week or more, depending on host cell type, transfection, and screening effectiveness. 

4) Pick 5-10 resistant clones and transfer them to 35 mm cell culture plates, and culture them in a drug-containing screening medium for 7 days. 

5) Replace with fresh medium without drugs for culture.