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

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Answer

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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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 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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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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Can the Neomycin resistance gene found in your mammalian expression vectors also be used for kanamycin selection in E. coli?

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Answer

No, these vectors will not express kanamycin resistance in E. coli because the Neomycin resistance gene does not have a prokaryotic promoter. You cannot select transformants on kanamycin. This has been verified by streaking a glycerol stock of pcDNA™3.1 vector onto 3 plates: LB, LB + Amp, and LB + Kan. After incubation overnight at 37 degrees C, the cultures were observed to exhibit growth on LB and LB + Amp but no growth on LB + Kan.

Answer Id: E3474

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