Hello Rose Miller

Some facts that you should know about RAW264.7 cells while optimizing.

1. You could either use DMEM + 10% FBS or RPMI +10% FCS. Use of 2mM L-glutamine and Pen/strep is optional.

2. These cells are most suitable to culture in a T75 cm2 flask. Total volume in the flask should be around 10mL. The growth media should be refreshed every 2-3 days but no more than 4 days without causing significant cell death. The average doubling time of RAW264.7 cells is 15 hours.

3. When the cell confluency reaches around 60-70%, the cells need to be split. Aspirate all growth media by tilting flask to allow media to collect in a corner. Wash once with PBS (half of culture volume) by gently aspirating PBS into a corner, gently shaking the PBS around the flask and aspirate. Eject half of culture volume of trypsin directly onto the cells and incubate for 5 minutes at 37 degree C. Please note that RAW cells are quite adherent to tissue culture flask. So, if the trypsin does not immediately lift the cells, do not proceed to wait longer as this risks the viability of cells. Eject (half of culture volume) warm media directly onto the cells. Gently use the cell lifter and scrape cells off the flask. These cells do not lyse easily with a cell lifter. Collect the cell suspension from flask. Spin for 8-10 minutes at 1500 rpm.

4. Resuspend in fresh media. It is important to triturate repeatedly to allow for a single-cell suspension. Seed cells into a new flask with dilution and place back into the incubator. 1:3, 1:4 or 1:5 dilutions are all acceptable. Please note that greater the dilution, the longer the cells will take to reach confluence. Do not heavily dilute as cells seeded with extremely low confluence exhibit impaired growth and proliferation.

Hope this information will help!

Good Luck!