I'm attempting to assess the degree of DNA damage caused by treatment with Zeocin (150micrograms/ mL) by utilizing the gamma H2AX assay. I create a 25mL stock of zeocin in DMEM and then place on my cells for 1h30minutes (i place plain DMEM media on my untreated cells), after which i fix, wash 3x in pbs, permeabilize with 0.5% triton X, wash 3x in pbs, block in 5% FBS for 1 hour and then incubate with the gamma h2ax primary over night.
But the issue when I image is that i'm seeing a lot of foci in my Heks not treated with zeocin. Do Heks have this degree of damage naturally or am I doing something wrong?
Hello
HEK293 is not an ideal cell line if would want to study any biological function. These cells have deregulated pRb/p53 pathways, and so any biological function related to these pathways will be affected, especially apoptosis.
Best.
Hi,
yH2Ax foci should be found in small percentage of cells. I also found it in untreated HeLa, U2OS and TCC SUP cell lines both using Immunofluorescence and WB. If Zeocin creates double-stranded DNA breaks yH2Ax activity should be increased after treatment. Is that what you see or is it the same as control cells (in % of cell with foci/total cells)?
Mateo Glumac i know that a few foci should be visible in the untreated hek cells but I see almost the same amount of foci in my untreated cells vs my treated cells and I’m not sure where the DNA damage is coming from
Zeocin is a weak dsb inducing compound (quite indirect and slow); when used as a selection marker, cells take weeks to die. We used etoposide in the past: quite potent, at least in transformed cell types; you could consider that as a positive control (see Article Topoisomerase IIα-dependent induction of a persistent DNA da...
). The gold standard remains gamma radiation, where the relationship of dsd breaks to dose are well defined. And yes, gH2AX foci are constantly forming, in any cell type due to spontaneous breaks etc..but there shouldn't be dozens of it.Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Fixatives