5 replies to this topic

[Piersgb]

I'm working on a mouse model system that involves looking at granulocyte function. I completely understand why not many people work or do anything with mouse blood due to the small quantities involved but I am trying to separate out lymphocytes from whole blood. Has anybody done anything like this before?

We already have Percoll and Ficoll-Paque Plus to carry out density sedimentation, but I do think that these have been designed for human blood samples. Does anybody know density/osmolarity values for mouse blood cells? I could then adjust the density of my gradients to something suitable for the mouse blood cells. Or are there any other easier techniques out there?

Thanks for reading!

[casandra]

Piersgb, on Jul 22 2010, 07:45 AM, said:

I'm working on a mouse model system that involves looking at granulocyte function. I completely understand why not many people work or do anything with mouse blood due to the small quantities involved but I am trying to separate out lymphocytes from whole blood. Has anybody done anything like this before?

We already have Percoll and Ficoll-Paque Plus to carry out density sedimentation, but I do think that these have been designed for human blood samples. Does anybody know density/osmolarity values for mouse blood cells? I could then adjust the density of my gradients to something suitable for the mouse blood cells. Or are there any other easier techniques out there?

Thanks for reading!

Hi Piersgb,

Welcome to the forums…. Have you already tried this:

A simple method for the isolation of murine peripheral blood lymphocytes
by Chia and Harris  J Immunol. Methods, 19, 169-172(1978)


- simple enough and just using Ficoll-Hypaque….good luck….

[Piersgb]

Ah, lovely! Thanks for the pointer. Though, I'm looking to get granulocytes ideally! No idea why I wrote lymphocytes. My bad.

So if anybody has any ideas of a way that I could get mouse granulocytes, to whatever degree - I'm not looking for massive purity, that doesn't involve some big expensive commercial kit I'd be very happy! I just can't really see any literature on this out there. Maybe I'll be the first?!

[casandra]

Piersgb, on Jul 22 2010, 12:23 PM, said:

Ah, lovely! Thanks for the pointer. Though, I'm looking to get granulocytes ideally! No idea why I wrote lymphocytes. My bad.

So if anybody has any ideas of a way that I could get mouse granulocytes, to whatever degree - I'm not looking for massive purity, that doesn't involve some big expensive commercial kit I'd be very happy! I just can't really see any literature on this out there. Maybe I'll be the first?!

hmmm...you shldn't mix up your whites .....anyways,....I found one which uses discontinuous percoll...3 layers I think and it's cheaper than Ficoll....btw, aren't you going to pool your samples?

Edited by casandra, 22 July 2010 - 08:31 AM.

[Piersgb]

casandra, on Jul 22 2010, 04:53 PM, said:

...

A simple method for the isolation of murine peripheral blood lymphocytes
by Chia and Harris  J Immunol. Methods, 19, 169-172(1978)
...

I also can't gain access to this paper. Are you able to attach a copy on a post anyway so that I could have a look at it?

This isolation is just a inital look-see. I'm looking at culturing mouse bone marrow cells to eosinophils and want to compare these cultured eosinophil to peripheral blood eosinophils. So if there is some contamination of other cell types I'm not too bothered right now. Just as long as I can detect PB eosinophils in the isolation!

Many thanks.

Edited by Piersgb, 22 July 2010 - 08:36 AM.

[casandra]

Piersgb, on Jul 22 2010, 12:34 PM, said:

casandra, on Jul 22 2010, 04:53 PM, said:

...

A simple method for the isolation of murine peripheral blood lymphocytes
by Chia and Harris  J Immunol. Methods, 19, 169-172(1978)
...

I also can't gain access to this paper. Are you able to attach a copy on a post anyway so that I could have a look at it?

This isolation is just a inital look-see. I'm looking at culturing mouse bone marrow cells to eosinophils and want to compare these cultured eosinophil to peripheral blood eosinophils. So if there is some contamination of other cell types I'm not too bothered right now. Just as long as I can detect PB eosinophils in the isolation!

Many thanks.

I will try to attach a copy....fyi, I'm the resident tech idiot ...or I'll send it by pm...whichever... and you're working on eosinophils? neutrophil contamination might be your bigger problem....let me see if I can do this...