I am doing an experiment with a large number or large TC dishes culturing HCT116 cells. Trypsin detachment was somewhat inconsistent in my hands with these large dishes (245 mm). I'm wondering if anyone has had success scraping this cell line and what consequences have been observed? Does scraping kill a large % of these cells? Can the cells still be counted after scraping using an automated cell counter (TC20, biorad)?
Jesse,
If you have some plates to practice on, I suggest you try it -- attempt scraping with some variant tools and techniques and inspect under a microscope with a live/dead assay.
What was the nature of the inconsistency with trypsin? It should be gentler than mechanical detachment if applied uniformly (enough depth in the dish) followed by prompt trypsin inactivation.
A relatively gentle mechanical method, which you might combine with trypsin, would be to agitate the plate for some fraction of a minute by holding it on top of a shaker such as a Vortex Genie. I used to do this (without trypsin) to harvest cells that were preparing to divide.
Good luck,
Doug MacDonald
Those are good ideas. Thanks!
I found out that I was using 0.05% trypsin instead of what I usually use without problems, 0.25% trypsin. I think I want to try scraping still anyway.
Any advice about what the best cell scrapers are? someone told me that they make some that are like windshield wipers and some that are very stiff.
I never use trypsin for cell detachment as it affects the viability and morphology of the cells. Accutase is a new and alternative to trypsin, it is from plant origion and not affecting on cell viability even if you incubate more than 10 minutes. I have also another idea that is my modification in cell culture, you can use PBS-BSA-EDTA containing 0.01% triton X-100. This is a very safe and very good for cell detachment. TQ
Thanks for your comments. In the end I scraped them in cold PBS and they needed to be triturated by pipetting to break up clumps. Counting without trituration was too variable.
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