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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!
- Grow cells almost to confluence.
- Remove growth media from cells.
- Aliquot 200ul cells to a 96 well plate for testing.
- 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.
- Observe the cells for the normal passage time prior
to analysis. For best results use duplicate plates
harvested on successive days.
- 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.
- 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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