Posted 17 June 2013 - 09:31 PM
it is my first time to use NIH 3t3 cells for the formation of native Extracellular matrix
I have some questions?
at first: I read some protocol about maintaining it Calf serum with high glucose DMEM.
But I find another website suggest 10 % FBS with DMEM (not high glucose)?
usually in our lab we use heat inactivated FBS, so I just thaw my cells in 10% heat inactivated FBS in DMEM.
I will try to see if it will effect Extra cellular matrix secretion or not but do u have any suggestion??
The second thing, they said never leave it till become 100% confluent, but I really left it, yesterday it was almost 70%, but I said leave it till tomorrow as I wanted to make alot of stock?
so today it was almost 100 % confluent??
how does this affect my cells.
Please if u have further comments regarding this type of cell, kindly share it with me.
Posted 18 June 2013 - 01:34 AM
In general for cell culture you want to ensure that the cells you are working with are as similar to the parent line as possible - one way to do this is to prevent them from undergoing selection pressures like the ability to grow well at confluence or low density - hence the 70% upper limit and split ratios you will have seen discussed. In practice, it seems to be uncommon for people to worry too much about this sort of thing, so you will probably be fine with your 3t3s as these are are a well established transformed cell line. However, many publishers are now asking that people validate their cell lines before accepting publications so major changes in cell behaviour will be noticed.
Posted 28 June 2013 - 02:25 AM
I agree with alot of what bob1 contributed. I have a couple of extra points:-
a) NIH3T3 cells are mouse fibroblastic cell line. You state " Usually in our lab we use heat inactivated FBS".......WHY
Heat inactivated was used years ago to reduce adventisious agents and compliment (please refer to the link below)
The most important information from the link is that heat inactivation WILL adversely affect the efficacy of your FBS i.e. its ability to support cell growth. More importantly is WHERE the FBS comes from i.e. ITS COUNTRY OF ORIGIN. New Zealand (NZ) serum is the most expensive FBS in the world. We batch test this and have seen in our experiments that the cells are "healthier" in NZ FBS.
Alot of the commercially available immortised cell lines WILL grow in most cell culture medias. You mentioned high and low concentration DMEM...these are just 2 variations of DMEM....there are very many others commercially available. The most important thing with DMEM is that you should always check if the varaition of DMEM has the original formulation i.e. the original DMEM requires you to grow the cells in 10% CO2 and NOT 5% CO2
The most important thing is to use the exact same conditions in all your experiments ONCE you have optimised all conditions i.e. FBS/Media/Plastics etc.
I hope you find this useful