I'm doing a cell count, in a 3D structure, to monitor cell proliferation at different time points: 1, 3, 7, 14, & 21 days. I'm looking for the best method to break down a PEGDA gel to be able to access to cell nucleus to preform a DNA assay. However, breaking down the gel network seems to be a challenge. I've done several methods, freezing at -80 c˚/dry, centrifuge, Ultrasonic cleaner bath and eventually I'm trying to use Tris-EDTA buffer solution 100×. I'm wondering, what is the best method to break down a 3D gel network since the PEGDA may not be a reversible gel?
AE
Hey Alarbi,
I´m not so familiar with PEGDA but if you use MMP-sites in your gel - for cell migration - you can try collagenase to break the structure down.
I recently did such a study with PEGDA gels encapsulating NIH3T3 cells. I would first break them in a microcentrifuge tube containing 0.1% Triton-x manually with a pipitte tip, followed by freezing in -80C. After thawing I would probe sonicate them on ice. This high intensity sonication breaks them good and I got very good results for the ensuing Picogreen assay for nuclear DNA quantifaction.
Hello Martin,
Thank you for your answer. Yeah I was thinking to use collagenase to breakdown the 3D structure.
Alarbi
Hello Atanu,
Thank you for your time. I have tried the same method in the past, however, I probe sonicated the samples in a hot water instead of ice. I am wondering why would you use ice? what was your time point during the experiment? I mean did it work with you during day 1, 7 and 14. Were you able to get good results during these time points?
Thank you
Alarbi
Alarbi
Hot water would denature DNA. While doing DNA work, it needs to be kept as cold as possible; sonication itself will heat up your sample enough to destroy all DNA and your precious time. So, try using ice. I also did not mention earlier that I also included a bath sonication (does not make much difference) prior to probe sonication, and i put lot ice in the bath regularly as well. While doing my Picogreen assay, I kept all the samples on ice as well. I did time points Day 1, 7, 14, 21 for my gels.
Thanks
Atanu
Yeah! the point is when I did sonication in water bath the water will heat up, my main goal was to destroyed the 3D structure. I'll definitely try prob sonication since it worked with you. How many seconds/minutes did you do sonication for each sample.
Alarbi
what about using live-dead assay to perform cell proliferation? Cause live-dead assay can dip into the gel, most of molecualr hydrogel use this method.
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