Antibiotic Kill Curve

Background

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.

Protocol for Kill Curve

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

What you need:

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 deg C 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.

Image 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 (Image 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 (Image 2).

Image 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

Recommended Concentrations

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 # 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

Important note: 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.

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Materials