RAW264.7 murine macrophage - (May/03/2009 )

hi all, I have a few questions related to RAW264.7

1. how to obtain the cell count more accurately? everytime i scratch the cell out and find that there are many cell clumps under the microscope

2. how many times can this cell line be passed? When I pass the cell in the same flask for 5~8 times, the cells seems starting to loss their aderence. Is that normal? Or is there anything I need to beware when I pass them?

Nrelo on May 4 2009, 12:46 PM said:

Are you gently aspirating the cells in and out of a pipette a few times to break up the clumps?

This will depend on the intended assay. I use RAW cells for TNF-a, and at times I’ve had to push out to 30 passages and there hasn’t been any obvious signs that their response has changed. (I use DMEM, 10% FBS)

Nrelo on May 4 2009, 03:46 AM said:

These cells should be grown in SUSPENSION culture....I use Techne stirrer platforms and bottles. If you do it this way there is no need for scraping or trypsinising the cells .....hence 99% viability. Also only trituration is require to get a single cell suspension......easy for counting!!!!!!!

How many passages you ask........Hundreds, my record is 216 passages. We look at iNOS enzyme induction using Interferon gamma and LPS......no difference in induction from passage 1 to 216.

HOWEVER that is only 1 cell parameter/marker....and it is of interest to us. OTHER MARKERS MAY NOT BE AS STABLE.

Hope this is useful

Regards

Rhombus