Raw 264.7 for 60-70% confluency tomorrow? - How many do i seed today? (Mar/06/2006 )

Hi,

Does anyone know how many Raw 264.7 cells I'd need to seed in a 24 well plate to achieve 60-70% confluence tomorrow?

Raw 264.7 cells grow VERY FAST. The doubling time is approximately 12 hours or less. I usually seed 1.0x10^5 cells per well with 1.0ml of medium. They are about 80-90% confluent after 24 hours.

By the way, do you use non-heat inactivated FBS or HI FBS for culturing RAW 264.7 ?

Another question: does the DMEM you use to grow RAW 264.7 cells contain sodium pyruvate? The media i currently use (4.5g/L glucose, 4mM glutamine, 1.5g/L sodium bicarb) does not contain any sodium pyruvate. My cells appear granular and "holey". I received a flask of very nice looking cells from another lab but after splitting just once, they already look like crap and viability is only 70-80%.

Im wondering if sodium pyruvate is required for healthy growth of these cells... I found several inflammatory labs dealing with RAW 264.7 cells have their cell culture protocols available online and the DMEM used all happen to contain some amount of sodium pyruvate, even though it is not stated as a requirement by ATCC.

any ideas??

Hi,

Cheers for that, I'd already plated them using a protocol I found on the net before I got your reply (if you're interested):

http://www.lipidmaps.org/protocols/PP0000000101.pdf

So I plated out 2x10(5) in 1 ml in a 24 well plate and this morning they look pretty good (around, 70-80% confluent)

About the media I use... I just use DMEM 4.5g/L glucose, 2mM L-glut, and 1% non-essential amino acids (as this is what our post doc recommended). There's no sodium pyruvate in my media and my cells seem to grow fine. I've also noticed that most other people use sodium pyruvate in their media but the lab I got these cells from didn't (I've only been working with these cells for about 2 wks so am learning as I go along for now)

Hope that helps