Hi,
I'm working on lung cancer cells, LLC1 (CRL-1642, ATCC) but I'm not familiar with it.
which media and what kind of FBS are good for LLC1? or are there anything important I need to know?
Many thanks.
Hello Tingting Wang
You need to know the following while culturing LLC1.
You may use DMEM + 2mM Glutamine + 10% Fetal Bovine Serum (FBS) which is heat inactivated.
The process of heat inactivation involves heating the FBS at 56 deg. C in a water bath for 30 mins. Please note that FBS has to be at a temperature of 56 deg. C for 30 mins. Immersing the FBS bottle in the water bath which is at 56 deg. C does not mean that the FBS bottle is at 56 deg. C. You may have to wait till the FBS in the bottle attains 56 deg. C, and then incubate for 30 mins.
For LLC1, subcultures are prepared by diluting the suspension 1:4 to 1:6. During subculturing, cells on the surface of the flask may be dislodged by aspirating several times with culture medium or by simply rinsing with 0.25% trypsin - 0.53 mM EDTA solution. During this process, be a little careful as cells may detach as aggregates and therefore may be difficult to count.
After 3-4 passages in culture, you may have to prepare stock of LLC1 to be stored in liquid nitrogen vapor phase for future use. The freezing medium that you will have to use is complete growth medium supplemented with 5% (v/v) DMSO. Store the cells in 1.5ml cryovials at 1 million cells/ml/per vial.
You will get more information on the ATCC website from the link below.
https://www.atcc.org/products/crl-1642
You may also want to refer to the image of healthy LLC1 cells in culture. Please refer to the link below.
https://www.culturecollections.org.uk/media/51886/90020104-ll2-llc1-images.pdf
Best.
Malcolm Nobre Thank you so much. The company suggests using non-heat inactived FBS and medium with less concentration of NaHCO3 may be better for LLC1. But in some papers, they just mentioned DMEM containing 10% FBS, 1% Pen/Strep, different from what the company suggests. Thus I was wondering.
Tingting Wang, you may follow the company’s instructions as this would be the proper way to go about. As far as heat inactivation of FBS is concerned, it is basically done to inactivate the complement system for immunoassays and it has also been reported to inactivate other undetermined inhibitors of cell growth in culture. Using non-heat inactivated FBS should not make much of a difference.
If you are well-versed in tissue culture techniques, and have never got contamination in your previous cultures, you need not add 1% Pen/Strep in complete DMEM.
- Badges
- Science method