Antibiotic Kill Curve

What is an antibiotic kill curve?

Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time. This is a crucial step before using a selection antibiotic to kill untransfected cells and generate stable cell lines. Since each mammalian cell line differs in antibiotic sensitivity, it is always recommended to perform separate kill curve experiments for different cell lines and antibiotics.

Recommended Concentrations of Antibiotics for a Kill Curve

The table below indicates the concentration ranges for commonly used selection antibiotics that may be adopted in dose response experiments.

Table 1 Recommended Concentrations of Selection Antibiotics

Selection Antibiotic	Product No.	Conc. Range
Actinomycin D	A9415	5 – 10 µg/mL
Amphotericin B	A9528 A2411	0.25 to 2.50 mg/mL
Amphotericin B solution	A2942	0.25 to 2.50 mg/mL
Bleomycin sulfate	B8416 203408-M	10 – 50 µg/mL
Carbenicillin	C3416	100 – 1000 µg/mL
Chloramphenicol	C3175 C1919	5 – 50 µg/mL
G 418 sulfate salt	345810 345812	100 – 800 µg/mL
G 418 disulfate salt	A1720 G1279	100 – 800 µg/mL
G 418 disulfate salt solution	G8168	100 – 800 µg/mL
G 418 solution	G418-RO	50 -1000 µg/mL
Gentamicin sulfate	G1264	50 – 1000 µg/mL
Gentamicin solution	G1272 G1397 G1522 345815	50 – 1000 µg/mL
Hygromycin B	H3274 H9773 400050	200 – 800 µg/mL
Hygromycin B solution	10843555001 400052 400053	200 – 800 µg/mL
Kanamycin sulfate	K1377 10106801001 420411	100 – 1000 µg/mL
Kanamycin solution	K0254 K0129	100 – 1000 µg/mL
Mitomycin C	M4287 M7949 10107409001	10 – 100 µg/mL
Mycophenolic acid	M3536	10 – 100 µg/mL
Neomycin trisulfate	N6386 N3144	10 – 100 µg/mL
Neomycin solution	N1142 480100	10 – 100 µg/mL
Paromomycin sulfate	P8692 P5057	100 – 1000 µg/mL
Puromycin	P8833	10 – 100 µg/mL
Puromycin solution	P9620	10 – 100 µg/mL
Rifampicin	R7382	10 – 50 µg/mL

The dose response may vary with the cell line being tested. The concentration ranges are only for guidance; a kill curve must be performed with every selection antibiotic when used for the first time or used on a new cell line.

Protocol for Kill Curve

The following protocol outlines the steps to generate kill curve for your cells.

What you need:

• Cell line of your interest in healthy condition
• Complete culture medium (medium + serum) recommended for your cell line
• Selection antibiotic of your interest

Steps:

• Harvest healthy adherent cells either by using trypsin or by gentle cell scraping.
• Dilute the cell suspension in complete medium and seed each well of 96-well plate so as that the final volume is 100 ul. The recommended cell density is ~50% on the day of treatment.
• Typical cell density before antibiotic treatment in a 24-well plate:
• 0.8 – 2.5 X 10⁵ cells/ml of adherent cells
• 2.5 – 4.5 X 10⁵ cells/ml of suspension cells
• Incubate at 37 ^oC overnight or till all the cells are well adjusted and healthy.

Note: Antibiotics are most effective when the cells are in active division; an optimum cell density is therefore recommended for most accurate dose response.

Figure 1. Schematic guide – selection antibiotic concentration.

• Replace the medium with fresh medium containing varying concentrations of selection antibiotic. Maintain each concentration in triplicates as indicated in the 96-well plate format (Figure 1).
• Replace the medium with antibiotic every 48 hours.
• Culture the cells for 7 – 10 days regularly observing the cells under light microscope. The duration of culture may be extended to 14 days depending on the cell line used.
• On day 10, determine the cell viability in each well either by MTT assay or by accurate cell counter.
• Choose a concentration of antibiotic that completely blocks the growth of cells. Use this concentration in all transfection experiments to select for resistant, transfected cells that can be further grown as stable cell lines (Figure 2).

Figure 2. Mock Antibiotic Kill Curve

Points to Remember

• Maintain appropriate controls or blanks:
  • wells containing only medium with antibiotic, no cells
  • wells containing medium and cells, no antibiotic
• Ensure the incubator is in perfect working condition to avoid evaporation of medium that might increase the concentration of antibiotic giving false readings

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