Raw 264.7 cells- Culture conditions? - (Apr/16/2007 )

Hi there,
My question is about how you are culturing Raw cells. Because I can never get them in good conditions I feel. They can not get more than 50% confluent, many of them start to float.
I would be glad to hear if you use
1. dishes or flasks?
2. RPMI or DMEM?
3. How often and in which ratio you split them.
Thank you for your answers.

In my lab, we grow Raw cells on dishes and use DMEM-F12 media.I split them when they reach almost 90% confluency.And I am using only cell scrappers when detaching the cells.I hope, these can help.Best luck.

Dear Clementine,

We have grown RAW cells for the past 15 years. You can grow them in either DMEM or RPMI depending upon your experimental protocol. We are interested in Nitric Oxide(NO) so we usually use DMEM, as it does not contain Nitrate, which is an oxidation product of NO.
You can grow them in !0%, 7.5% down to 5% FBS/FCS.
The best way for them to proliferate is to grow them in SUSPENSION culture. There are many advantages to this method:-
a) No TRYPSINISATION : thus you do not need to use PBS/Trypsin or culture media(to inhibit the trypsin). As you know RAW cells are difficult to trypsinise because they adhere to the TC plastic like glue.
Money saved on disposable TC plastic and other consumables: Once you have bought the borosilicate glass stirrer bottles, they can reused by Autoclaving them. Mine bottles have lasted 20 years so far.
c) YOUR TIME: the most important thing: subculture and cell preparation takes just minutes i.e. pour off cells and centrifuge, pour in fresh media.

THESE ARE THE EASIEST CELLS IN THE WORLD TO GROW. And for our studies on NO, the amount of iNOS we can produce from cells at P1 is the same as at P200....this is also useful if you want to compare experimental results from one year to the next.

Hope this is helpful

Rhombus

i am sorry that i've said i am using cell scrapper for suspension cells.sorry again.i must be sleepy when i was writing....No need to write anything, i think Rhombus has given the answer.

Dear Lazarus and Rhombus,
Thank you very much for your suggestions.
I will try that.
Cheers