I am planning to do primary macrophage cell culture. I couldn't decide what to use to remove cells from the well plate prior to RNA isolation procedures, for example we usually use trypsin for this. But in primary culture this can be somewhat harmful. What should I use?
Dear Cemre Kirşan ,
I usually apply the lysis buffer direct onto the plate, use a cell scraper to collect everything in the lower edge of the vessel and transfer the buffer into a Eppendorf tube.
As Sönke Weinert said, generally, RNA isolations are performed by adding your lysis buffer directly to the plates. I've seen some people trypsinize their cells and pellet before adding the lysis buffer, but that isn't necessary.
For suspension cells, you'll need to pellet the cells first to remove the media. and add your lysis buffer to the cell pellet.
For larger culture vessels (basically anything that isn't a multiwell plate), I recommend using a cell scraper to pull the lysate to one corner for transferring to a tube (as Sonke said).
Hello Cemre Kirşan
You may wash the cells with cold PBS (without calcium and magnesium), and leave the cells in cold PBS for 10-15 mins. Then use the cell scrapper and gently scrape them.
Alternatively, you may wash the cells with cold PBS (without calcium and magnesium), and leave the cells in 5mM EDTA on ice for 15-20 mins. Then with the help of the cell scrapper you may gently scrape them.
Best.
Hi Cemre Kirşan There are several methods that can be used to remove cells prior to RNA isolation in primary macrophage cell culture, including:
Regardless of the method used to remove the cells, it is important to carefully handle the cells during the process to avoid damaging the cells or compromising the quality of the RNA that will be isolated. After removing the cells, it is also important to immediately preserve the RNA to avoid degradation. This can be done by adding a RNA stabilizing reagent, such as RNAlater, or by immediately proceeding with the RNA isolation procedure.
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