8 replies to this topic

[bert]

hi all,

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?

many thanks!


bert


PS. i work with mouse bone marrow.

[Piersgb]

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!

[lab rat]

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%.

[bert]

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.

cheers,

bert

Edited by bert, 12 November 2010 - 06:21 AM.

[lab rat]

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 [salt] is lower in your suspension (e.g. PBS) and the cells come into contact with the higher [salt] Percoll during the layering, the cells will flatten and slide through the colloid.

[aris_c]

Hi all,

 

i am not sure if this topic is still active, but i also have a question.

I have almost established a culture of endothelial cells with some fibroblast contamination and i want to separate those two cell types.

Now i know that it can be done using Percoll, but the manual is a bit confusing and i get perticularly confused with the density specifications.

The endothelial cells should be separated, according to my reference, at 1.07 g/ml and the fibroblasts at 1.05-1.055.

Can anybody expain me how i can make those two specific densities?

 

Thank you all,

 

Aris

[mdfenko]

you can use this on-line calculator from ge lifesciences.

[aris_c]

Hi mdfenko,

 

thanks a lot for your response.

I am aware of the on-line calculator, but i wil be performing this kind of separation for the first time.

So, if i understand correctly i have to make the solutions for specific densities. But what about the osmolarity, will the solution with my prefered density be ok for my cells? will they survive? besides that, i only want to collect just one type of cells, so i  guess if i make just one density percoll, then i will only get the cells that i want layered. But how does this look like in real time? I will be pouring the DMEM where i have my cells in on top of this Percoll solution, then i will spin it and then what happens?

Do you have any experience with Percoll?

 

I appreciate you responce a lot.

 

Thanks again.

 

Aris

[mdfenko]

sorry, i haven't used percoll myself.

 

here is the instruction booklet from ge lifesciences (pdf download)

 

osmolarity should be controlled by the buffer you use.

Edited by mdfenko, 24 September 2014 - 05:47 AM.