Antibiotic Kill Curve: Definition and Protocol
What is an antibiotic kill curve?
The antibiotic kill curve is a dose-response assay used to select stable gene cell lines. When generating stable cell lines in mammalian cells, if the expression system carries resistance genes, stably transfected/transduced cells can be selected by adding antibiotic drugs to the medium. Because different types of mammalian cells differ in their sensitivity to antibiotics, the minimum antibiotic concentration required to kill untransfected cells must be determined by a separate kill curve experiment. During the experiment, the antibiotic concentration was continuously increased in the cell culture medium until all cells died. Faster-growing cells need to be cultured for seven to ten days, and slow-growing cells may need to undergo selection for up to 15 days, depending on their growth rate. Antibiotic screening was then performed using the lowest antibiotic concentration sufficient to kill all cells, and resistant transfected cells were screened for further culture into stable cell lines.
Recommended antibiotic concentrations for kill curve
Here's a list of the most common selection antibiotics and their normal ranges of concentration in mammalian cell lines:
| Catalog | Product Name | Category | Inquiry | Concentration range |
|---|---|---|---|---|
| BBF-03755 | Actinomycin D | Antibiotics | Inquiry | 5–10 µg/mL |
| BBF-00698 | Carbenicillin | Antibiotics | Inquiry | 100–1000 µg/mL |
| BBF-00635 | Chloramphenicol | Antibiotics | Inquiry | 5–50 µg/mL |
| BBF-03869 | Chloramphenicol succinate | Antibiotics | Inquiry | 5–50 µg/mL |
| BBF-03972 | Chloramphenicol succinate sodium | Antibiotics | Inquiry | 5–50 µg/mL |
| BBF-03794 | Geneticin sulfate | Antibiotics | Inquiry | 100-800 μg/mL |
| BBF-05841 | Gentamicin Sulfate | Antibiotics | Inquiry | 50–1000 µg/mL |
| BBF-01729 | Hygromycin B | Antibiotics | Inquiry | 200–800 µg/mL |
| BBF-02559 | Mitomycin C | Antibiotics | Inquiry | 10–100 µg/mL |
| BBF-02567 | Mycophenolic Acid | Antibiotics | Inquiry | 25 µg/mL |
| BBF-05608 | Neomycin trisulfate | Antibiotics | Inquiry | 10–100 µg/mL |
| BBF-05845 | Neomycin sulfate | Antibiotics | Inquiry | 10–100 µg/mL |
| BBF-04562 | Rifampicin | Antibiotics | Inquiry | 10–50 µg/mL |
| BBF-02567 | Mycophenolic Acid | Antibiotics | Inquiry | 25 µg/mL |
| BBF-04117 | Nourseothricin sulfate | Antibiotics | Inquiry | 75–100 µg/mL |
| BBF-03812 | Paromomycin sulfate | Antibiotics | Inquiry | 100–1000 µg/mL |
| BBF-04103 | Puromycin dihydrochloride | Antibiotics | Inquiry | 10–100 µg/mL |
| BBF-04164 | Kanamycin acid disulfate | Antibiotics | Inquiry | 100–1000 µg/mL |
| BBF-04549 | Kanamycin B Sulfate | Antibiotics | Inquiry | 100–1000 µg/mL |
| BBF-04214 | Bleomycin sulfate | Antibiotics | Inquiry | 10–50 µg/mL |
| BBF-00701 | Carfecillin sodium | Antibiotics | Inquiry | 100–1000 µg/mL |
A number of factors influence the correct amount of antibiotics to be administered, such as:
Growth rate of cell: In fast-dividing cells, less can be needed, because antibiotics work at cell division.
Type of antibiotic: Intensity and mechanism of action (e.g., inhibition of protein synthesis) varies between antibiotics.
Density and confluency of cell: The antibiotics work best at certain cell densities (30-50% confluence at start).
Antibiotic kill curve protocol
Materials
Target cell line for health status
Complete media required by cells (medium + serum)
Antibiotic
Steps
Harvest healthy adherent cells by trypsin or gentle scraping.
The cell suspension was diluted with a complete medium and inoculated into each hole of the 96-well plate with a final volume of 100 ul. The recommended cell density on the day of treatment is about 50%.
Typical cell density in 24-well plate before antibiotic treatment:
- 0.8-2.5×105 cells/mL adherent cells.
- 2.5-4.5×105 cells/mL suspension cells.
Incubate at 37 °C overnight or until all cells are well adjusted and healthy.
Note: Cells in the active division state are the most sensitive to antibiotics. Therefore, the appropriate cell density is recommended to achieve the most accurate dose response.
Complete media were replaced with fresh media containing different concentrations of screened antibiotics, with at least 3 controls per concentration.
Replace the antibiotic-containing medium every 48 hours.
The cells were cultured for 7-10 days and observed under a light microscope at regular intervals. For slower growing cells, the culture time can be extended to 14 days.
On the 10th day of culture, the cell viability of each pore was measured by MTT assay or accurate cell counter. Obtain the lowest antibiotic concentration that completely kills cell growth.
In subsequent transfection experiments, the lowest antibiotic concentration obtained by the experiment was used to screen for tolerant transfected cells that could further grow into stable cell lines.
Tips for antibiotic kill curve
Ensure all laboratory instruments and reagents are clean and disinfected to prevent contamination.
Use healthy cells with logarithmic growth to ensure high cell viability.
Verify that the cells are not contaminated and record the number of passages of the cells, as too many passages may affect cell characteristics.
Ensure that the control group and the experimental group are consistent on other variables (such as culture conditions).
Pay attention to the spacing of the concentration gradient to avoid the span being too large or too small.
Keep appropriate contrast or blank:
The pores contained only antibiotic-containing media and no cells.
A hole containing culture medium and cells, free of antibiotics.
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