Saturday, 4 December 2010

GGS LIVE - Foci kinetics

Yo, Yo, Yo Biofreak readers!!

Method: Foci kinetics.

About: Foci kinetics assay is performed in order to assess foci formation and resolution of particular protein under specific conditions.

What: Kinetics of gamma-H2AX foci formation after ionizing radiation in wild-type and mutant cells.

In our study case experiment we will investigate gamma-H2AX (histone modification) foci formation and resolution after treatment with ionizing radiation. Ionizing radiation cause DNA double strand breaks.
In response to such DNA damage H2AX histone is phosphorylated (called gamma-H2AX in phosphorylated state) to facilitate double strand break repair.
Briefly, DT40 cells were treated with IR (5 Gy), harvested and analysed by immunoflurescence at different times post-IR treatment (for immunofluorescence tutorial visit GGS LIVE - immunofluorescence). Pictures below represent cells stained for gamma-H2AX harvested at different times post-IR treatment.

As you see on the images above there is not much of signal from gamma-H2AX in untreated cells (there is small number of spontaneous DNA damage in unchallanged cells - red arrow heads indicate representative gamma-H2AX foci). After induction of double strand breaks with IR treatment H2AX histone is robustly phosphorylated (15 min timepoint) and this modification is removed with ongoing DNA repair. At this stage we have to quantify our results. We can eitehr simply score number of the foci per cell or score number of cells with more than X foci of gamma-H2AX. Plot below represents such quantification in which cells with 6 or more gamma-H2AX foci were scored as positive.

From the quantification plot you can see that both cell lines induce gamma-H2AX foci fomration with the same kinetics, indicating that this process is not affected in the mutant cell line. When we look at gamma-H2AX foci resolution, four hours post-IR treatment 50% of wild-type cells has resolved gamm-H2AX foci, where mutant cells need another 4 hours to accomplish the same task. Such results indicate that mutant cell line might have problems with repairing DNA damage caused by IR treatment.

I hope you enjoyed:)



1 comment:

  1. A study on gamma-H2AX (histone modification)is very effective & informative.Your detailed information never i had seen any where.Thanks a lot.