RAW 264.7 cell line - (Jun/08/2013 )
I want to use RAW 264.7 cell Line. But still have some unclear problems regarding that. I will use cell line by borrowing from another lab. Those are telling me that this cell line is semi-suspension cell line, so I don't need to use Trypsin and PBS. Though when I go through web I found it is firmly attaching to the walls and need to use cell scraper.
Also I wanted to know about the serum. Some are using newborn calf serum. though i found some are using fetal bovine serum.As that FBS too expensive it would be better that if I can use newborn Calf serum
Please clarify this for me.
Thank you very much in advance.
I found no difference between serum types in dose response/ NO experiments with RAW’s although the growth is about 20% slower with newborn serum. I’d recommend that you splash out on at least one bottle of fcs to determine if your experiments are affected by the growth media.
Check the archives; I remember a discussion about 3 years ago on whether RAW’s out to be treated as attached or suspended.
Don't trypsinize RAW cells. They are adherent unless they are overgrown. Just remove the media. Add a bit of fresh (Approx 5ml for T-150) and scrape. Trypsin is a good way to make those cells very unhappy. To grow I used DMEM with 10% FCS.
Thank u very much for the replies. They were helpful to me.Also I found some cells are firmly adherent to the surface and those adherent cells had different shapes. Some of them had characteristic shape of the macrophages and some had other shapes.
However thank u very much for the replies.
I worked with RAW 264.7 cells in the late 1980's as a source of inducible nitric oxide synthase (iNOS). This enzyme was used to screen potential iNOS inhibitor compounds made synthesised by our chemists. My initial take was to optimise the induction conditions in order to produce the most iNOS for the screen. This involved:
Choosing the lipolysaccharide to use: Salmonella typhimurium (10ug/ml)
Choosing the dose of interferon gamma to use (50 units/ml)
Choosing the optimised stirring speed (cells were grown and induced in suspension culture)
However one of the most important optimisation factors was thge SERUM. We used exclusively New Zealand origin FBS/FCS as in our hands this serum gave us 50-70% more induction of iNOS than using other origin FBS/FCS.
The RAW cells are an adherent cell line and stick to tissue culture plastic like superglue. Thus we grew our cells in Techne Stirrer bottles that are made out of borosilicate glass.....this means that the cells DO NOT stick to the glass.
Hope some of this is useful