Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching
作者:
BattagliaMassimo,
PozziDeleana,
CrimaldiSettimio,
ParasassiTiziana,
期刊:
Biotechnic&Histochemistry
(Taylor Available online 1994)
卷期:
Volume 69,
issue 3
页码: 152-156
ISSN:1052-0295
年代: 1994
DOI:10.3109/10520299409106277
出版商: Taylor&Francis
关键词: fluorescence;photobleaching;bisbenzimide;Hoechst 33258;Fluorochromes;p-phenylenediamine;n-propyl gallate,1,4-diazabicyclo(2,2,2)octane;mycoplasma;cell culture
数据来源: Taylor
摘要:
DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules,p-phenylenediamine (PPD),n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.
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