ANTIBIOTIC TITRATION KILL CURVE

A "kill curve" is used to determine the concentration of antibiotics required to kill cells within a
period of time. The minimum antibiotic concentration to use is the lowest concentration that kills
100% of the cells in 1-4 days from the start of antibiotic selection. Selection vs. maintenance is
different. Selection is about twice that of maintenance.
G418 (Geneticin) is a broad-spectrum antibiotic that will select mammalian cells expressing the
neomycin protein (encoded by the neomycin gene). Neomycin, kanamycin and G418 are all
similar compounds known as aminoglycosides that can be inhibited by the product of the neo
gene, neomycin phosphotransferase. G418 is specific for the eukaryotic ribosome while the
others more specifically inhibit the bacterial organelle. Dissolve G418 sulfate in H 2O (100 mg/ml),
filter sterilize and store in small sterile 1 ml aliquots at -20C.
G418. In duplicate, use the following concentrations: 0, 100, 300, 500, 700 and 1000 g/ml
antibiotic in complete medium (such as DMEM + 10%(v/v) FBS).
Puromycin inhibits the growth of a wide range of eukaryotic and prokaryotic cells by interfering
with protein synthesis. It allows the selection of cells expressing the pac gene. Dissolve
puromycin dihydrochloride in DI H 2O (10 mg/ml), filter sterilize and store in 100 l aliquots at
-20C.
Puromycin. In duplicate, use the following concentrations: 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,
8.0, 9.0, 10.0 g/ml antibiotic in complete medium (such as DMEM + 10%(v/v) FBS).
1. Generate a cell suspension (in complete growth medium without antibiotic) from adherent
cells, which will be used for transfection or transduction. Cells may be harvested either by cell
scraping or by trypsinization.
2. Using the same cell density to be used in subsequent transfection or transduction procedures,
plate cells and culture overnight under appropriate conditions (e.g., 37 C with 5% CO 2). Cells are
usually plated in 24 well-plates with 500 L of complete medium/well.

For adherent cells: Plate cells at a density of 0.83.0 x 10 5 cells/ml.

For suspension cells: Plate cells at a density of 2.55.0 x 10 5 cells/ml.

3. Most cell types should be 80% confluent prior to adding the selection antibiotic. Replace
complete growth medium with selection medium supplemented with a range of antibiotic
concentrations. Include untreated control cells with growth medium only (no selective antibiotic
added).
4. Monitor the cells daily using a microscope and observe the percentage of surviving cells.
5. Culture for 14 days. Approximately every 2-3 days replace medium with freshly prepared
selection medium containing the range of antibiotic concentrations being tested.
6. Use the lowest concentration of your antibiotic that begins to give massive cell death in 3 days
and kills all the cells within two weeks. A general starting point is usually 400 mg/ml G418 for
HeLa cells. In mammalian cells the optimal level of puromycin is typically around 1 mg/ml.
7. Proceed with stable cell line generation using the concentrations determined in step 6. Cells
transfected with a plasmid harboring the puromycin-N-acetyl-transferase (pac) gene should be
grown in complete growth medium for 4872 hours post-transfection before selection antibiotic is
applied.
Suderinti sia nuoroda su emiau esaniu protokolu
https://www.mirusbio.com/applications/stable-cell-line-generation

Transfection and Drug Selection

1. Grow cells to ~80% confluence in complete medium and transfect your plasmid
with appropriate method, for example LipofectAmine Plus (Invitrogen) is a good
method for HeLa cells.
2. After 24-48 hours of transfection, cells are split to 1:10, 1:20 or 1:50 into (2) 15 cm
plates containing 25 ml of DMEM + 10% FBS + appropriate concentration of drug.
Leftovers of transfected cells can be plated in 10cm plates containing 10 ml media.
If you use Tet-OFF system, add 5 g/ml of tetracycline or 1 g/ml of doxycycline
to shut off the protein expression.
3. Observe cell growth in every 2-3 days and change medium with selection drug
every week or more if necessary. After 24 weeks, isolated colonies should begin
to appear.
Isolation of Drug Resistant Clones
4. Melt 1% low melting agarose by microwave and incubate in a water bath at 37C
until cool down below 37C. Mix 10 ml of 1% agarose and 10 ml of 2x DMEM +
20% FBS (and doxycycline if necessary) and pour into 15 cm plate (media
removed). Leave the plate at RT for less than 30 min. Put the plate into a CO2
incubator for refreshment.
5. Mark large, healthy and well separated colonies and put colony
separator (see figure) directly on surface of soft-agarose. Apply
gentle pressure to top of separator to prevent movement.
6. Add 100l of trypsin-EDTA, pipeting several times gently to
penetrate the soft-agarose surrounding the isolated colony and
incubate at RT.
7. Place trypsin treated colony into one well of a 48-well plate containing 1 ml of
DMEM + 10% FBS + drug.
II.C.5
8. Split clones that reach ~80% confluence into one 12-well plate and one 6-well
plate. Use one plate for checking protein expression/induction. Check the protein
expression of each clone by its fluorescent intensity under a fluorescent microscope
or by Western blotting.
9. Split only expression positive clones to 10 cm plate and store in liquid N2.
10. For sub-cloning, re-plate about 100 cell per plate. For example, immediately after
splitting, take 10-100 l of culture and re-plate in 10 cm plate.
11. Repeat steps 5-11. You may pick 6 colonies to check for protein expression.