RAW 264.7 cell self-activation is often a result of environmental factors such as endotoxin contamination, cell culture conditions, or passage number. By carefully selecting reagents, controlling culture conditions, and monitoring for contamination, one can minimize this issue. If spontaneous activation persists despite these measures, the researcher may need to adjust the experimental design or switch to another macrophage model.

Hello Juha Park

You may consider the following.

1. RAW 264.7 cells will self-activate when grown to high density before splitting. Keeping them at lower density should stop self-activation. Cells should be split when the confluency is between 70 and 80%.

2. During subculture, do not use trypsin for cell detachment. RAW 264.7 cells are sensitive to external stimuli. Trypsin can activate these cells. Trypsin treatment may greatly influence the cell membrane protein composition and reactivity of the cell to media components. Instead, you may use EDTA, manual scraping, or a mixture of the two to harvest RAW 264.7 cells.

3. Test your cells for mycoplasma contamination.

4. Have you changed the lot/brand of FBS used in the media? The serum can have lot-to-lot effects.

FYI: The standard media used for the growth and maintenance of RAW 264.7 cells is DMEM. Though RPMI-1640 has been used to maintain RAW 264.7 cells, there are a few differences in the composition that you should make a note of.

For instance, RPMI-1640 lacks the high level of glucose present in DMEM (4.5g/L) which in turn could affect the response of RAW 264.7 to external stimuli.

You may want to refer to the paper attached below for more information.

https://digitalshowcase.lynchburg.edu/studentshowcase/2024/oral_presentations/28/

Best.