Liver cancer primary cell culture - urgent!Thanks (Jul/13/2008 )
I need to discuss the method on culturing the primary liver cancer cells. However, I have ever done any cell cultue before.
I have somes questions about the following protocol
the livers were removed and perfused first with Buffer A (10 mM Hepes, pH 7.4, in calcium/magnesium-free Hanks' buffered saline solution (HBSS), gentamycin sulfate (1 mg/ml medium), and 0.5 mM EGTA) for 10 min (3 ml/min),
followed by Buffer B (Buffer A without EGTA, supplemented with 5 mM CaCl2 and 0.2 mg/ml collagenase for an additional 10 min.
1. What is the function of Hepes?
2. What is HBSS? function? why cannot add calcium ad Mg?
3. What is gentamycin sulfate and its function?
4. What is EGTA and its function?
5. What is Buffer B, cannot add EGTA but add CaCl2?
1)Hepes is a pH buffer
2)HBSS is hank's balanced salt solution, it is a normal saline solution that mimics the body's salt levels. EGTA will chelate the Ca and Mg
4)metal ion chelator
5)see answer about EGTA.
Also, I really really suggest getting someone experienced in cell culture to show you the ropes before attempting such delicate work as primary cell culture. You need to be familiar with working under aseptic conditions and familiar with the hood and equipment needed so as to not contaminate your precious primary samples.
Get out I. R. Freshney's Culture of Animal Cells: A Manual of Basic Technique, it is really good and has protocols and techniques for primary culture of many different cell types.