I want to assess the effect of radiation on normal human astrocytes, which are co-cultured with glioblastoma cells. However, a clonogenic assay is not suitable for this since astrocytes do not form colonies. I have searched the web and found a hybrid clonogenic assay where the authors counted astrocytes and glioblastoma cells 5 days after radiation (Sarcar et al. Targeting Radiation-Induced G2 Checkpoint Activation with the Wee-1 Inhibitor MK-1775 in Glioblastoma Cell Lines, 2011). Does anyone have any other ideas on how to assess the effects of radiation damage in non-colony forming cells?
Hello Steven,
If cell growth or death still actual for dose of interest then possible to estimate changes in metabolic activity (MTT, resasurin based methods etc). The estimation of level of oxidative stress biochemical markers also could be informative (TBARS, 4-HNE, 8-oxoguanine, carbonil adducts) as well as production pro/anti-inflammatory cytokines or apoptotic, autophagy and death processes (g-H2AX, comet assay, DNA fragmentation etc).
Hope these ideas could be helpful!
As suggested above, you can measure the cell proliferation by cell counting or MTT assays; besides, you can also see irradiation-induced DNA damage, such as gamma-H2AX foci or Rad51 foci formation in cells, and COMET tail assay, etc; the check-point factors or mediators for cell cycles can be detected as well.
Hope this information are helpful for you!
Dear Daniil and Han,
Thank you for the good advice. I have considered these options before, but the problem is that these are all assays that assess the influence on a short-term basis. Since radiotherapy primarily exerts long-time effects, detecting DSBs or HR might not be representable anymore then. Cell counting might be a valid option, but it does not take loss-of-reproduction potential into account.
Hi Steven, colony formation assays are usually used to evaluate the stem-like properties of cells, and another common way to assess the stem-like properties is to track the PKH26 labeled cells. PKH26 is also mostly used for the identification of quiescent or slowly dividing cells based on dye dilution. You can monitor the PKH26 dye intensity via flow cytometry to measure the division or proliferation in your cells w/ or w/o radiation. Moreover, PKH26 staining is very stable, and can be used for the long-term experiment.
Great! If you need more then you have to take a look on Dr. Soile Tapio papers.
Find one attached bellow and link to new version not available on RG yet.
Hope you will find suitable biomarkers and share your success with us!
Article Ionizing radiation biomarkers for potential use in epidemiol...
Article Ionizing radiation biomarkers in epidemiological studies – An update
Dear Steven
Post-irradiation, astrocytes, I think, show nuclear changes.
Therefore it would be a good idea to evaluate multinucleation, binucleation, micronucleation, etc. This is very easy to do in cultures too, a little bit of patience is neede; no sophisticated instruments.If you have the facility, you can try various FCM parameters. Please see my papers on real-time cytology work in oral squamous cell cancers
Narayanan
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells
- Cancer Stem Cells