Hello everyone,
I am splitting 264.7 raw macrophages using cell scraper. I have noticed that the viability is not high enough to do an experiment. When I switched to 0.05% Trypsin, I noticed an increase in the cell viability even though the ATCC recommend scraping cells. Any idea?
Thank you,
Muhannad: Cell scraper detached cells by mechanical means, while trypsin-EDTA by enzymatic means digesting the ECM. The loss of ECM makes cells to produce new ones for the attachment to plate surface thus promoting their growth. Moreover, enzyme digestion ensures full separation of individual cells, thus increased their viability. I never use scraper for this and other reasons. Good luck.
We are using RAW264.7 cells extensively in our lab and never used cell scraper or Trypsin at the time of splitting. We always remove cells by pipetting medium (in which cells are already growing). For example, if you are growing cells in 10cm dish using 10ml medium then (at the time of detaching) take blue pipette and suck 1000ul medium by tilting the plate towards you and apply that medium on attached cells (keeping the plate angle around 45 degree). The pressure will allow cells to detach from the surface; and keep doing it by rotating the plate unless all cells have been detached. It will keep the viability without activating the cells.
Do you use "non-tissue culture treated" flasks? It is possible to culture RAW macrophages on Petri dish or even in Shott glass bottle. In our case non-TC flasks improved viability of scraped RAW264.7 a lot.
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