Which of your antibiotics (Geneticin, Zeocin, Hygromycin B, Blasticidin, and Puromycin) can be used together for stable selection in mammalian cells?
All of our antibiotics (Geneticin, Zeocin, Hygromycin B, Blasticidin, and Puromycin) can be used together for making multiple stable cell lines. However, kill curves will need to be performed for each combination of antibiotics since sensitivity to a given antibiotic tends to increase when combined with other antibiotics.
What are the recommended concentrations of antibiotics to use for selection in prokaryotes and eukaryotes?
For best results, optimal concentrations for selection should be determined empirically in each unique experiment through dose response curves. However, to get a general idea of concentrations that have worked for individual cell types, please click on the following url: http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/selection.html or type in “Selection Antibiotics” into our main search on www.thermofisher.com.
In contrast to Geneticin (G418)-induced cell death, cells treated with Zeocin do not always detach and float when they die. Is this typical?
It is true that a percentage of non-resistant mammalian cells do not round-up from the plate upon Zeocin selection as would be seen with G418 or Hygromycin selection. However, one should see some very characteristic morphological changes occurring in those cells that are not resistant. These cells that stick to the culture dish typically display a vast increase in size. This could be best described as being similar to the effects of cytomegalovirus infecting permissive cells. The shape of these cells may also change; taking on an "alien" shape. On close examination of the non-resistant cells, the researcher should observe a distinct breakdown of both the nuclear and plasma membranes. Even though the "cells" are still attached to the plate, they should have the appearance of many holes in these membranes. Also, before the breakdown of the membranes, one can observe open areas in the cytoplasm of the cells that appear to be large, empty vesicles. Although not confirmed, this may be explained by a breakdown of the endoplasmic reticulum and Golgi apparatus, or other scaffolding proteins. Eventually, these "cells" will completely breakdown so that only "strings" of protein are left.
In contrast, Zeocin resistant cells should continue to divide at a regular interval to form distinct clumps of cells, or colonies. There should not be a distinct change in morphology, which can be compared to cells not under selection with Zeocin. It is these colonies of actively dividing cells that contain the resistance gene and are expressing it actively.
If there is concern about the dead cells sticking to the plate, one may do the following to eliminate them: Treat the plate for a couple of minutes with trypsin/versene. Both the healthy resistant cells and the dead cells will dislodge from the plate. The cells can then be replated (without Zeocin selection) and the healthy cells will attach again while the dead ones will not. After a couple of hours when the healthy cells have attached to the substrate again, Zeocin can be added back to the medium.
What is the mode of action on the following antibiotics: Blasticidin, Geneticin (G418), Hygromycin, and Zeocin?
Blasticidin: Nucleoside Inhibits protein synthesis in prokaryotic and eukaryotic cells by interfering with peptidyl transfer reaction of protein synthesis, causing early termination of translation.
Geneticin (G418): Aminoglycoside Blocks protein synthesis in mammalian cells by interfering with ribosomal function.
Hygromycin: Aminocyclitol Inhibits protein synthesis by disrupting translocation and promoting mistranslation.
Zeocin: Intercalates with DNA and cleaves it.
How can I decontaminate my cultures?
When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination.
1. Determine if the contamination is bacteria, fungus, mycoplasma, or yeast. Read more here to view characteristics of each contaminant.
2. Isolate the contaminated culture from other cell lines.
3. Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters.
4. Antibiotics and antimycotics at high concentrations can be toxic to some cell lines. Therefore, perform a dose-response test to determine the level at which an antibiotic or antimycotic becomes toxic. This is particularly important when using an antimycotic such as Gibco Fungizone reagent or an antibiotic such as tylosin.
The following is a suggested procedure for determining toxicity levels and decontaminating cultures:
1. Dissociate, count, and dilute the cells in antibiotic-free media. Dilute the cells to the concentration used for regular cell passage.
2. Dispense the cell suspension into a multiwell culture plate or several small flasks. Add the antibiotic of choice to each well in a range of concentrations. For example, we suggest the following concentrations for Gibco Fungizone reagent: 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 µg/mL.
3. Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding.
4. When the toxic antibiotic level has been determined, culture the cells for two to three passages using the antibiotic at a concentration one- to two-fold lower than the toxic concentration.
5. Culture the cells for one passage in antibiotic-free media.
6. Repeat step 4.
7. Culture the cells in antibiotic-free medium for four to six passages to determine if the contamination has been eliminated.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
