how to make Percoll solutions?! - (Oct/26/2010 )
the percoll manual gives me the creeps really. i dont wanna study physics before being able to generate a percoll solution...
so is there anybody out there who could give me a "for dummies" procedure of how to generate this isotonic stock solution and then from that to get a 60% percoll solution?
PS. i work with mouse bone marrow.
I work with mouse bone marrow too! I've also been playing with Percoll and from studying the manual I've come up with the following:
You first need to make a 1.5M solution of NaCl. Then to 9 parts Percoll (from the bottle) you add 1 part 1.5M NaCl. This is to make Percoll isotonic, and from then on the manual refers to this solution as Stock Isotonic Percoll (SIP) as 100% Percoll (confusing, I know!).
To make further dilutions of Percoll, you dilute with 0.15M NaCl.
To get a 60% Percoll solution you want to have a final solution that is 60% SIP : 40% 0.15M NaCl. So to make 10mls of 60% Percoll, mix 6ml SIP with 4ml 0.15M NaCl.
Hope that helps!
We used hank's balanced salt solution, but same principle:
Mix 10 ml HBSS in 90 ml 100% Percoll (now 90%). This is the isotonic Percoll, like Piersgb said.
Dilute 60 ml isotonic Percoll with 40 ml 1X HBSS (plain).
The nomenclature in the manual confuses everyone at first, so I just gave these directions to the students to make their lives easier. The actual % percoll is around 54%.
many thanks guys!!!
and sorry for the late answer... was busy with other stuff (the percoll thing is more of a sidekick project of mine).
what you say is reassuring, really. and i mean in two ways.
first, i dont seem to be the only one who feels a bit stupid when reading this horrible manual.
second, i allways did it as Piersgb described, but then sometimes the seperation didnt work, which means it wasnt due to the percoll but probably the sample itself. so i gotta work on handling the cells more gentle i guess.
anyways, i recently tried again and it worked out. btw, i ran ficoll in parallel and it worked as well but gave less sharp bands and less recovery than percoll.
Glad to hear it Bert. I thought of one more thing: it helps to make sure the cells are suspended in a solution of the same osmolarity as the Percoll. If the