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HOME > Protocols > Cell Biology > Cell Culture Protocols > Protocol for Antibiotic Titration for Cell Culture

Protocol for Antibiotic Titration for Cell Culture

Many antibiotics can also be toxic to cells in culture. It is recommended to titrate your antibiotics in your growth media with your specific cell line (or primary cells) prior to experiment. It is also important to point out that an antibiotic may affect your readout in certain experiments and I normally don't use them except when culture primary cells. I may also culture cells in antibiotics when they are obtained from an outside source that I do not trust!

  1. Grow cells almost to confluence.
  2. Remove growth media from cells.
  3. Aliquot 200ul cells to a 96 well plate for testing.
  4. Apply the antibiotic concentration to the wells, keeping the concentration constant within each row of 8. Make sure to have a row that contains no antibiotic.
  5. Observe the cells for the normal passage time prior to analysis. For best results use duplicate plates harvested on successive days.
  6. For analysis it is suggested to use methylene blue, trypan blue or propidium iodide/ethidium bromide staining or some other method to enumerate live and dead cells.
  7. Graph the live dead ratio and select the highest concentration in which there is little or no effect on the growth of your cell line.

Note: It is recommended that this titration be repeated if any of the lot numbers of any of the components of your media change (serum, media, antibiotics). The release assays for the manufacturer may allow for some variation in the function of your assay. This is good advice for any rigorous cell culture experiment.

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