Blasticidin S HCl (10 mg/mL), 20 mL - FAQs

哺乳动物细胞的稳转筛选过程中可一起使用哪些种类的抗生素（Geneticin，Zeocin，潮霉素B，杀稻瘟素和嘌呤霉素）？

我们所有的抗生素（Geneticin，Zeocin，潮霉素B，杀稻瘟素和嘌呤霉素）均可共用于多重稳转细胞系的建立。不过，需要针对每一种抗生素组合来获取杀菌曲线，因为当与其他抗生素联用时，细胞对某一特定抗生素的灵敏性会出现增加的趋势。

我该如何对我的培养物去污染？

当不可替代的培养物被污染时，研究人员可能会试图控制或消除污染。

1.用户需要确定污染的来源是细菌、真菌、支原体，还是酵母。请点击此处(https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/biological-contamination/bacterial-contamination.h%E2%84%A2l)阅读更多信息，以了解每一种污染的特性。
2.把受污染的培养物跟其他细胞系进行隔离。
3.使用一款实验室消毒剂清洁培养箱和层流柜，并检查HEPA过滤器。
4.高浓度的抗生素和抗真菌剂可能对一些细胞系有毒性。因此，需进行剂量效应测试来确定何种浓度水平的抗生素或抗真菌会造成毒性。这一操作对于使用Gibco Fungizone一类的抗真菌剂或泰乐菌素一类的抗生素尤其重要。

下列操作为我们确定毒性水平和对培养物去污染的推荐步骤：

1.对细胞进行分离，计数, 使用不含抗生素的培养基稀释将细胞稀释至常规传代的浓度。
2.将细胞悬液分入多孔培养板或几个小培养瓶中。向每一培养孔中添加不同浓度的特定抗生素。举例来说，我们推荐以如下浓度测试Gibco Fungizone试剂:0.25，0.50，1.0，2.0，4.0和8.0 µg/mL。
3.每日观察细胞脱落，出现空泡，融汇度降低，细胞变圆一类的毒性效应。
4.一旦确定了抗生素的毒性浓度水平，就可使用比毒性浓度低一至两倍的抗生素浓度来培养细胞两至三代。
5.在不含抗生素的培养基中培养一代。
6.重复步骤4。
7.在不含抗生素的培养基中培养细胞四至六代，以确定污染是否成功被消除。

你们提供哪些抗生素来帮助用户控制或减少细胞培养中的污染情况？

请访问如下页面(https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/antibiotics.html)浏览我们提供的细胞培养相关的抗生素产品。

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.

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.

What is the optimal pH of low salt LB for LB + blasticidin plates?

We recommend a pH of 7 or less and half the normal amount of NaCl in your LB media or plates.

See the following paper for details on optimal conditions: Yamaguchi et al (1965) Inhibition of Protein Synthesis by Blasticidin S. Journal of Biochemistry (Tokyo) Volume 57: pp 667-677.

How long can Blasticidin be stored at 4 degrees C after thawing? Does the unused portion have to be discarded after thawing?

Blasticidin is stable for 6 months when stored at 4 degrees C. Discard remaining material after this time.

What solvent can be used to dissolve Blasticidin?

Blasticidin can be dissolved in water or acetic acid. Make stocks of 5-10 mg/ml.

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.

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

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).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.