Greetings
Can anyone suggest a technique for harvesting adherent fibroblasts from 12 well plate with minimal strain to the cells?
Dear Basant
Depends o what type of downstream processing and investigation you intend to do. Trypsin treatment is the standard protocol. You can also scrape the cells using a rubber spatula , while keeping the plate on ice [of course after removing the media]
I can agree with colleague Mr. Ravi Pottathil that it depends on type of downstream processing and investigation Basant intend to do. Trypsin treatment is the standard protocol. You can also scrape the cells using a rubber spatula , while keeping the plate on ice after removing the media.
Depending on the type of fibroblast, you might try exposure to an EDTA or EGTA solution. Sometimes chelation of Ca2+ is sufficient to release the cells.
Thanks all for your contributions.
Actually, i'm planning to extract mRNA from the cells for RT-PCR experiment.
In this context, which is less harmful:
harvesting cells using trypsin or trypsin-EDTA?
In that case, skip the cell harvesting, lyse the cells in situ, and go straight to RT-PCT. Check out the link below - no company affiliation. I expect that other companies also offer similar extraction kits.
http://www.lifetechnologies.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction/cells-to-ct-kits.html#how
I agree with Mr Bruce, just straight way lyse the cells and look fr the proper precautions
mmmmmm ... the problem is: i intended performing the pcr in other lab .. i may travel with my samples ... so, i thought that transporting cells is much better than RNA as it is quite unstable!
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