How can I decontaminate my cultures?

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Answer

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.

Answer Id: E11916

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What is the mode of action on the following antibiotics: Blasticidin, Geneticin™ (G418), Hygromycin, and Zeocin™?

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Answer

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.

Answer Id: E3693

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Which of Invitrogen™'s antibiotics (Geneticin™, Zeocin™, Hygromycin, and Blasticidin) can be used together for selection in mammalian cells?

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Answer

All of Invitrogen™'s antibiotics (Geneticin, Zeocin™, Hygromycin, and Blasticidin) can be used together. However, kill curves will need to be determined for each combination of drugs since sensitivity to a given drug tends to increase when combined with other drugs.
Note: Puromycin is of the same family as Blasticidin so Puromycin and Blasticidin may not be compatible.

Answer Id: E3948

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What antibiotics do you offer to help control or eliminate cell culture contamination?

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Please view the following page to browse the cell culture antibiotics we offer (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/antibiotics.html).

Answer Id: E11861

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In contrast to Geneticin™ (G418)-induced cell death, cells treated with Zeocin™ do not always detach and float when they die. Is this typical?

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Answer

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.

Answer Id: E3699

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How do I prepare a Geneticin™ solution from the powder?

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Answer

The specific activity of Geneticin™ varies from lot to lot. To create a stock solution of Geneticin™, obtain the potency from the label or Certificate of Analysis. Make a concentrated stock at 50 mg/ml active weight in sterile dI water, buffered salt solution, culture medium, or 100mM HEPES buffer. Note: Geneticin™ in water is acidic and adding it directly to the medium will often cause an immediate pH change, which will be plainly visible as a color change in medium containing phenol red. If the medium is well-buffered, the pH will return to normal in a matter of minutes to a few hours. If the pH change is of concern, you may wish to make the stock solution in buffered solution such as 100mM HEPES.

Answer Id: E2986

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Which of your antibiotics (Geneticin™, Zeocin™, Hygromycin B, Blasticidin, and Puromycin) can be used together for stable selection in mammalian cells?

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Answer

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.

Answer Id: E9151

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What are the recommended concentrations of antibiotics to use for selection in prokaryotes and eukaryotes?

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Answer

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.

Answer Id: E3701

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Can Neomycin be used in mammalian selection?  Can Neomycin be used instead of Kanamycin in bacterial selection?

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Answer

No, Neomycin is toxic to mammalian cells. It also causes irreversible damage to kidneys and other organs. Geneticin™ (aka G418 Sulfate) is a less toxic and very effective alternative for selection in mammalian cells.  Neomycin can be used in bacterial selection, but Kanamycin is the preferred drug to use because of Neomycin's toxicity.

Answer Id: E3949

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What dose of Geneticin™ (G418) should I use for selection?

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Answer

Sensitivity to G418 differs markedly depending on cell type, cell growth medium, and the serum supplement used. Experimentally determine the proper concentration for selection by setting up a dose response curve. Use concentrations between 100 ug/ml and 2 mg/ml G418 in medium, at 200 ug/ml increments. For more information, see the product insert.

Answer Id: E2985

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